Insulin and Growth Factor Signaling

Effects on Drug-Metabolizing Enzymes
  • Sang K. Kim
  • Kimberley J. Woodcroft
  • Raymond F. Novak
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Expression of drug-metabolizing enzymes may be altered in response to development, aging, gender, genetics, nutrition, pregnancy, disease states such as diabetes, long-term alcohol consumption, and inflammation, and by xenobiotics. Although the mechanisms by which xenobiotics regulate drug-metabolizing enzymes have been intensively studied, relatively little is known regarding the cellular mechanisms by which drug-metabolizing enzymes are regulated in response to endogenous factors such as hormones and growth factors. The first major section of the chapter defines the major insulin- and growth factor-mediated signaling pathways implicated in regulating drug-metabolizing enzyme expression, including those involving mitogen-activated protein and phosphatidyl inositol 3-kinases, small G proteins, and phosphatases. The second major section of the chapter presents a summary and evaluation of methods for determination of the role and function of signaling pathways that are involved in the regulation of drug-metabolizing enzyme gene and protein expression, including methods for determination of kinase activity and phosphorylation, the use of kinase inhibitors and dominant-negative protein kinase constructs, and the application of new RNA interference methods.

Key Words

Cytochrome P450 extracellular signal-regulated kinase glutathione S-transferase G proteins green fluorescent protein insulin insulin receptor jun N-terminal kinase kinase assays kinase inhibitors mammalian target of rapamycin (mTOR) microsomal epoxide hydrolase mitogen-activated protein kinase pathway p38 mitogen-activated protein kinase p70 ribosomal protein S6 kinase p70 S6 kinase phosphatase and tensin homologue deleted on chromosome ten pleckstrin homology primary culture protein kinases protein phosphatases rat hepatocytes receptor tyrosine kinase small interfering RNA (siRNA) small temporal RNA stress-activated protein kinases sulfotransferase UDP-glucuronosyltransferase 

1 Introduction

The body is equipped with several mechanisms to ensure that xenobiotics do not accumulate. Polar molecules are often poorly absorbed into the body, but when absorbed they are readily eliminated via the kidneys. In contrast, nonpolar molecules are a special problem because of their affinity for membranes. Increasing the polarity of small, nonpolar molecules through metabolism generally promotes the excretion of the metabolites. Xenobiotic biotransformation is the principal mechanism for maintaining homeostasis during exposure of organisms to small foreign molecules and occurs predominantly in the liver, although it also occurs in nasal mucosa, intestine, kidneys, lungs, placenta, and skin.

The reactions catalyzed by xenobiotic-, or drug-metabolizing enzymes are generally divided into two groups, phase I and phase II. Phase I reactions introduce a functional group that increases hydrophilicity and are mediated by cytochrome P450 (CYP), flavin-containing monooxygenase, xanthine oxidase, prostaglandin H synthase, amine oxidase, alcohol dehydrogenase, aldehyde dehydrogenase, epoxide hydrolase, and esterase. Phase II reactions include glucuronidation, sulfation, methylation, glutathione conjugation, and amino acid conjugation. In general, these reactions, with the exception of methylation, result in a large increase in xenobiotic hydrophilicity.

It is generally recognized that the expression of drug-metabolizing enzymes may be altered in response to development, aging, gender, genetic factors, nutrition, pregnancy, and pathophysiological conditions such as diabetes, long-term alcohol consumption, inflammation, and protein-calorie malnutrition. The expression may also be altered by xenobiotics. Although the mechanisms by which xenobiotics regulate drug-metabolizing enzymes have been intensively studied, relatively less is known regarding the cellular mechanisms by which drug-metabolizing enzymes are regulated in response to endogenous factors such as hormones and growth factors. Recent findings, however, have revealed that hormones and growth factors play an important role in the regulation of drug-metabolizing enzyme expression. Furthermore, the cellular signaling pathways involved in hormone- and growth factor-mediated regulation of drug-metabolizing enzymes are currently being studied.

Pathophysiological conditions such as diabetes, fasting, obesity, and long-term alcohol consumption result in increased expression of several hepatic enzymes, including CYP1A1, 2B, 3A, 4A, 2E1, and bilirubin UDP-glucurono-syltransferase (UGT1A1), whereas decreased expression of CYP2C11, microsomal epoxide hydrolase (mEH) and sulfotransferases (SULTs), such as hydroxysteroid SULT-a (SULT2A1) and aryl SULT IV (SULT1A1), has been reported (Table 1) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). On the other hand, studies of the expression and activity of glutathione S-transferase (GST) during diabetes are inconclusive, with both increased and decreased GST expression being reported in vivo (5,18, 19, 20, 21). The reason for this discrepancy remains unknown. However, it may, in part, be associated with competing factors in vivo and with variations in oxidative stress, usually observed in diabetes. It has been reported that transcriptional activation of some GST genes may be associated with the change in the redox state in conjunction with oxidative stress (22,23).
Table 1

Effect of Diabetes, Insulin and Glucagon on Drug-Metabolizing Enzyme Expression and/or Activity



Restored by insulin
















Marginally increased(35)



Decreased (9,12)











Yes (5)














Increase(18,20,21)/ Decrease (5,19)




GST alpha





GST pi





GST mu





ND, Not determined.

Because these pathophysiological states all result in altered hormone (insulin, glucagon, growth hormone) secretion, these hormones may be etiologic factors affecting the expression of hepatic drug-metabolizing enzymes. It has been reported that insulin or growth hormone administration to chemically induced or spontaneously diabetic rats restores drug-metabolizing enzyme activity and expression to control values (Table 1) (5,6,9,24, 25, 26, 27, 28, 29). Our laboratory and others have demonstrated that the activity and/or expression of hepatic drug-metabolizing enzymes such as CYP2B, CYP2E1, CYP2C11, CYP2A5, GST alpha class, GST pi class, UGT, and mEH are regulated by insulin and glucagon (Table 1) (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40). These results indicate that changes in drug-metabolizing enzyme mRNA or protein levels observed in pathophysiological conditions may be attributed to alterations in these hormone levels. Thus, it is of interest to identify which cellular signaling pathways are involved in regulating the expression of these genes in response to hormones.

In addition to altered expression of drug-metabolizing enzymes by patho-physiologic conditions, the pattern of drug-metabolizing enzyme expression is changed during development and aging and occurs in an organ-, sex-, and species-specific manner. Such observations also suggest that a cellular or organ context regulates the expression of drug-metabolizing enzymes. Growth factors, including epidermal growth factor (EGF) and hepatocyte growth factor (HGF), have a role in regulating drug-metabolizing enzyme gene expression. HGF results in decreased CYP2C11 expression in primary cultured rat hepatocytes (41) and decreased CYP1A1/2, 2A6, 2B6, and 2E1 activities in primary cultured human hepatocytes (42). In primary cultured rat hepatocytes, addition of EGF suppresses constitutive and xenobiotic-inducible CYP expression including CYP2C11, CYP2C12, CYP1A1, CYP2B1/2 (43, 44, 45) and CYP2E1 (Woodcroft et al., unpublished data). EGF has also been reported to increase alpha and pi class GST expression (46,47).

Our laboratory has demonstrated that the expression of CYP2E1 is suppressed by insulin (Fig. 1) and enhanced by glucagon in primary cultured rat hepatocytes (35, 36, 37). In contrast, the expression of alpha-class GSTs (Fig. 2) and mEH is enhanced by insulin and decreased by glucagon (33,40). Treatment of cells with glucagon also inhibits the expression of pi-class GST (33). Phos-phatidylinositol 3-kinase (PI3K) and p70 ribosomal protein S6 kinase (p70 S6 kinase) appear to play a central role in mediating the suppression of CYP2E1 expression (48) as well as enhancement of mEH (40) and alpha-class GSTs (Kim, Woodcroft and Novak, unpublished data). The regulation of CYP2E1 expression by insulin does not involve extracellular signal-regulated kinases (ERK1/2) or p38 mitogen activated protein kinase (p38 MAPK) (48). This is in contrast to the findings for insulin-mediated expression of mEH, which appears to involve p38 MAPK (40). Furthermore, our results implicate cAMP and protein kinase A (PKA) in mediating the effects of glucagon on CYP2E1, GSTs, and mEH expression (33,37,40).
Fig. 1.

Changes in CYP2E1 mRNA levels in primary cultured rat hepatocytes maintained in culture medium in the presence of various concentrations of insulin for 96 h. Values are shown as a percentage of the 1000 nM insulin value (100%). Columns and cross bars represent means ± SEM of Northern blot band densities of six to nine preparations of total RNA. Values were normalized for loading using 7S RNA. Significantly different than 1000 nM insulin-treated cells, p<0.05. (Reprinted from ref. 35. Copyright 1997, with permission from Elsevier.)

Fig. 2.

Immunoblot analysis of GSTA1/2 (A) and GSTA3/5 (B) protein levels in primary cultured rat hepatocytes maintained in culture in the presence of various concentrations of insulin for 3 d. Values are shown as a percentage of the GST levels monitored in hepatocytes cultured in the absence of insulin (100%). Data are means ± SD of immunoblot band densities of three preparations of cell lysates. ✻✻,✻✻✻Significantly different than levels monitored in hepatocytes cultured in the absence of insulin, p<0.01 or p<0.001, respectively. (Reprinted from ref. 33 with permission.)

In this chapter, an overview of the signaling pathways implicated in regulating drug-metabolizing enzyme expression in response to insulin and growth factors is described along with the methods for identifying which signaling pathways and components are involved in mediating this regulation.

2 Insulin-and Growth Factor-Mediated Signaling Pathways

The numerous and varied biological functions of insulin and growth factors are mediated by their corresponding cell surface receptors. The insulin receptor and growth factor receptors belong to the large family of receptor tyrosine kinase (RTK) cell surface receptors possessing intrinsic tyrosine kinase activity. After binding of their corresponding agonist, these receptors undergo autophosphorylation of tyrosine residues in the cytoplasmic domain and initiate a complex series of intracellular signaling cascades that ultimately result in diverse cellular responses.

Insulin and growth factors stimulate the recruitment of a family of lipid kinases known as class I PI3Ks to the plasma membrane. There, PI3Ks phosphorylate the glycerophospholipid phosphatidylinositol (PI) 4,5-bisphosphate at the D-3 position of the inositol ring, converting it to PI 3,4,5-triphosphate (PI[3,4,5]P3). Recent evidence indicates that serine/threonine protein kinase Akt/PKB (protein kinase B), atypical protein kinase C (PKC), and p70 S6 kinase mediate many of the downstream events controlled by PI3K.

Insulin and growth factors also lead to activation of mitogen activated protein kinase (MAPK) signaling pathways. On recruitment and activation via phosphorylated RTKs, the small guanosine triphosphatase protein Ras activates Raf, which leads to a phosphorylation signaling cascade involving activation of the MAPKs. RTK signaling is regulated not only by a cascade of phosphorylation via protein kinases, but also by dephosphorylation via tyrosine and serine/threonine phosphatases and lipid phosphatases. A simplified scheme of insulin receptor signaling as discussed in this chapter is illustrated in Fig. 3.
Fig. 3.

Insulin-mediated signaling pathways.

2.1 Insulin Receptor

The insulin receptor is a transmembrane heterotetramer consisting of two α- (extracellular; 135 kDa) and two β- (transmembrane; 95 kDa) subunits linked by disulfide bonds (49). During transport to the cell surface, a single high-molecular-weight proreceptor is proteolytically cleaved at a tetrabasic amino acid sequence (arginine-lysine-arginine-arginine) located at the junction of the α- and β-subunits, and oligosaccharide chains are added at specific sites of glycosylation. Interactions between the two α-subunits, and between the a and β subunit, are stabilized by disulfide bridges (50,51).

Unoccupied α-subunits on the cell membrane surface inhibit the intrinsic tyrosine kinase activity of the β-subunit and may be viewed as a regulatory subunit of the catalytic intracellular subunit (52). The β-subunit is composed of a short extracellular domain, a transmembrane domain, and a cytoplasmic domain that possesses intrinsic tyrosine kinase activity. The cytoplasmic domain contains the ATP binding site and autophosphorylation sites. Binding of insulin to the α-subunits of the receptor induces conformational changes leading to activation of the RTK activity, resulting in transphosphorylation of the β-subunits and endocytic internalization of the receptor via clathrin-coated vesicles (51). Some of the tyrosine-phosphorylated residues of the β-subunits of the receptor present binding sites for the subsequent recruitment of signaling molecules. The insulin receptor uses a family of soluble adaptors or scaffolding molecules, such as insulin receptor substrates (IRSs 1–4) and Shc molecules, to initiate its signaling cascade through other effectors.

Whereas the IRSs lack intrinsic catalytic activity, they have pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, and multiple phosphorylation motifs. The PH domains are globular protein domains of about 100–120 amino acids found in more than 150 proteins to date. PH domains are primarily lipid-binding modules, although they are also involved in mediating protein–protein interactions. The PTB domain of IRS binds to phosphorylated NPXP motifs in the insulin receptor and are subsequently phosphorylated on multiple tyrosine residues by the activated insulin receptor kinase. Following phosphorylation, IRS attracts and binds additional effector molecules to the vicinity of the receptor, thereby serving to increase the diversity of the signaling pathways initiated by the insulin receptor (53,54). The primary effector that binds to IRSs in response to insulin receptor activation is the lipid kinase PI3K that produces PI(3,4,5)P3 and subsequently activates Akt, PKC, and p70 S6 kinase (Fig. 3).

The adaptor protein Shc exists in p46, p52, and p66 isoforms and possesses Src homology-2 (SH2) and PTB domains and three tyrosine-phosphorylation sites. In response to extracellular signals, Shc is phosphorylated on tyrosine residues and binds the growth factor receptor binding protein 2 (Grb2), which is constitutively associated with the guanine nucleotide exchange factor Son of Sevenless (SOS) (55). Recruitment of the Grb2–SOS complex to the vicinity of Shc induces exchange of GDP to GTP on the membrane-bound GTPase Ras, thereby activating Ras. Activated Ras binds Raf and activates the serine/threonine Raf/MAPK kinase (MKK)/MAPK signaling pathway (Fig. 3) (56).

2.2 Growth Factor Receptor

The growth factor receptors are also members of a large family of cell surface receptors, including the EGF receptor; ErbB2, 3, and 4; and c-MET, which exhibit intrinsic protein tyrosine kinase activity. The EGF receptor is synthesized from a 1210-residue polypeptide precursor; after cleavage of the N-terminal sequence and glycosylation, the 1186-residue protein is inserted into the cell membrane (57). The glycosylated extracellular domain of the EGF receptor contains conserved cysteine-rich clusters that comprise the ligand-binding domain. Within the intracellular domain, the juxtamembrane region is required for feedback attenuation by PKC, followed by the tyrosine kinase domain and C-terminal regulatory tail. The C-terminal tail contains the tyrosine autophosphorylation sites and motifs for internalization and degradation of the receptor. Binding of EGF to the receptor results in receptor dimerization and autophosphorylation. The resulting conformational change creates specific docking sites for recruitment and activation of additional signaling proteins that contain SH2 and PTB domains, including Shc and Grb2. In addition, the EGF receptor can be phosphorylated by other kinases, such as PKC and Src kinase, which regulate the distribution and kinase activity of the EGF receptor (58,59). Following ligand binding and activation, EGF receptor dimers are recruited into clathrin-coated pits, which initiates a rapid endocytosis and degradation of the EGF receptor.

2.3 Pl3K/Akt/p70 S6 Kinase/Atypical PKCs

There are four major classes of PI3Ks, designated class I–IV; class I is also subdivided into Ia and Ib subsets. Class IV PI3Ks are not known to possess lipid kinase activity, but are serine/threonine kinases. The different classes of PI3Ks catalyze phosphorylation of the 3′-OH position of phosphatidyl myoinositol lipids, generating different 3′-phosphorylated lipid products that act as second messengers. Class Ia PI3Ks are primarily responsible for production of 3′-OH phosphoinositides in response to insulin and growth factors (60). Class Ia enzymes are dimers composed of a 110-kDa catalytic subunit that is associated nonconvalently to an 85- or 55-kDa regulatory subunit (Fig. 3). The catalytic subunit in subclass Ia is subdivided into p110 α-, β-, and δ. The regulatory subunit maintains the catalytic subunit in a low-activity state in quiescent cells and mediates its activation through interactions between SH2 domains of the regulatory subunit and phosphotyrosine residues of activated growth factor receptors or adaptor proteins, such as the IRSs (61). The single class Ib PI3K is the p110 γ catalytic subunit complexed with a p101 regulatory protein and mainly activated by heterotrimeric G protein-based signaling pathways. Direct binding of p110 γ to activated Ras plays an important role in the stimulation of PI3K in response to growth factor (62), but the physiological significance of this interaction in insulin-mediated PI3K signaling is not entirely clear.

Following the recruitment of PI3K to the plasma membrane, the lipid kinase phosphorylates the 3′-OH position of the inositol ring to generate PI(3,4,5)P3, PI(3,4)P2, and PI(3)P. The preferred substrate of class I PI3Ks appears to be PI(4,5)P2. These events occur within the first minute of insulin binding to its receptor and resulting lipid products then interact with a number of signaling proteins with PH domains, resulting in their membrane targeting and/or modulation of their enzyme activity.

The rapid increase in PI(3,4,5)P3 concentration in response to insulin activates several protein kinases, such as phosphatidylinositide-dependent kinase 1 (PDK1), Akt, PKC isoforms, and p70 S6 kinase (63, 64, 65). Among the PI(3,4,5)P3-dependent kinases, Akt has received much attention. Akt/PKB was identified as a protein kinase with a high degree of homology to PKA and PKC, and is the cellular homologue of the viral oncoprotein v-Akt. Akt is a 57-kDa serine/threonine kinase with a PH domain and the three known isoforms of Akt (Akt1, 2, 3) are widely expressed (66).

Akt exists in the cytoplasm of unstimulated cells in a low-activity conformation. The activation of Akt1 by insulin and growth factors is accompanied by its phosphorylation on threonine-308 in the kinase domain (T-loop) and serine-473 in the C-terminal regulatory domain (hydrophobic motif). Activation of Akt and phosphorylation of both these residues are abolished by pretreatment of cells with PI3K inhibitors such as wortmannin and LY294002 (67). After activation of PI3K, association of PI(3,4,5)P3 at the membrane brings Akt and PDK1 into proximity through their PH domains and facilitates phosphorylation of Akt at threonine-308 by PDK1 (65). The mechanism mediating serine-473 phosphorylation remains to be clarified.

p70 S6 kinase catalyzes the phosphorylation of the S6 protein, a component of the 40S subunit of eukaryotic ribosomes, and thus plays a role in protein synthesis (68,69). p70 S6 kinase participates in the translational control of mRNA transcripts that contain a polypyrimidine tract at their transcriptional start site. Although these transcripts represent only 100–200 genes, most of these transcripts encode components of the translational apparatus. The initial step in p70 S6 kinase activation appears to involve a phosphorylation-induced conformational change in the C-terminal domain, revealing additional phosphorylation sites. Subsequently, phosphorylation of the newly exposed sites (threonine 229, 389, and serine 371) occurs, which is dependent on both PI3K and the mammalian target of rapamycin (mTOR), based on wortmannin and rapamycin sensitivity, respectively.

Although expression of a constitutively membrane-anchored and active Akt variant induces the activation of p70 S6 kinase (70), Akt does not appear to represent the immediate upstream effector of p70 S6 kinase. Conus et al. (71) suggested that p70 S6 kinase activation could be achieved independent of Akt. Dufner et al. (72) demonstrated that a constitutively active wortmannin-resistant form of Akt was sufficient to induce glycogen synthase kinase-3 and eIF4E-binding protein 1 phosphorylation, but not phosphorylation and activation of p70 S6 kinase. The data suggest that p70 S6 kinase activation by membrane-targeted forms of Akt may be an artifact of membrane localization and that Akt resides on a parallel PI3K-dependent signaling pathway to that described for p70 S6 kinase.

Recent findings indicate that atypical PKC isoforms (ζ, rat) and (λ, mouse) serve as downstream effectors for PI3K (73). Increased activity of PKCζ/λ results from PDK1-dependent phosphorylation of the catalytic domain, via threonine-410 in rat PKCζ and threonine-411 in mouse PKCλ, followed by autophosphorylation of threonine-560 in rat PKCζ and threonine-563 in mouse PKCλ (64,74). PI(3,4,5)P3 may interact with the N-terminal lipid-binding domain of PKCζ to facilitate the interaction of threonine-410 with the catalytic site of PDK1 (74). PI(3,4,5)P3 also stimulates autophosphorylation of PKCζ and relieves the autoinhibition exerted by the N-terminal pseudosubstrate sequence on the C-terminal catalytic domain of PKCζ (74,75). Insulin-stimulated glucose transport and protein synthesis are dependent on PI3K/PKCζ activity (73,76). The latter is consistent with the observation that dominant-negative PKCζ antagonizes activation of p70 S6 kinase (77). However, it is not known whether PKCζ can directly phosphorylate p70 S6 kinase or which residue(s) is/are involved.

2.4 Ras/Raf/MEK/ERK

Many RTKs, including the insulin and growth factor receptors, are known to activate intracellular protein serine/threonine kinases, termed MAPKs, that phosphorylate various cellular targets in a proline-directed manner, including transcription factors and other kinases. The MAPK family consists of subfamilies with multiple members (Fig. 4): these include the ERK1/2, the Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), the p38 MAPKs and ERK5. Each MAPK is a member of a three-protein kinase cascade; a MAPK kinase kinase (MKKK) phosphorylates a MKK, which subsequently phosphorylates the MAPK. Of the various MAPKs, the ERK1/2 subfamily was the first to be characterized. The basic arrangement of the ERK signal cascade includes Ras, Raf (MKKK), MEK1/2 (MKK), and ERK1/2 (MAPK) (Fig. 4).
Fig. 4.

MAPK signaling cascades. (Adapted from website of Cell Signaling Technology.)

Mammalian cells contain three different Ras genes that give rise to four Ras small GTPases—H-Ras, N-Ras, KA-Ras and KB-Ras—which are key regulators of signal transduction pathways controlling cell proliferation, differentiation, survival, and apoptosis (78,79). In response to a great variety of extracellular stimuli, including hormones, growth factors, cell–extracellular matrix interactions, and oxidative stress, Ras proteins are activated through the GDP/GTP nucleotide exchange factor SOS, which induces the exchange of GDP for GTP, and thereby converts Ras to its active form. Ras cycles between the inactive GDP-bound and active GTP-bound states through the controlled activity of GTP nucleotide exchange factors and GTPase-activating proteins. After activation of insulin and growth factor receptors through agonist binding, the link between RTKs and Ras is provided by the GTP exchange factor SOS that exists in a complex with the adaptor protein Grb2 in the cytosol. Phosphorylated tyrosine residues in insulin and growth factor receptors are docking sites for Grb2. In addition, the interaction between Grb2/SOS and the receptors can be mediated by the adaptor protein Shc, which becomes tyrosine phosphorylated when recruited to the cytoplasmic domains of the activated receptors. This process brings SOS to the plasma membrane in close proximity to Ras, where it can promote GDP/GTP exchange. GTP-bound activated Ras recruits and activates three main classes of effector proteins, Raf kinases, PI3K, and RalGDS (62).

Three genes encode for the Raf family of serine/threonine kinases found in mammalian cells: A-Raf, B-Raf, and Raf-1 (c-Raf). The large majority of studies regarding the role of Raf in ERK activation have been performed with Raf-1. In resting cells, Raf-1 is located in the cytoplasm and is stabilized by a 14-3-3 scaffold protein dimer binding to phosphorylated serines 259 and 621, which are phosphorylated in resting cells (80). The binding of Raf to Ras and translocation to the plasma membrane can displace 14-3-3 from phosphoserine 259, which makes it accessible to dephosphorylation and activation by protein phosphatase 2A (PP2A) (81), although the role of dephosphorylation of serine-259 in Raf-1 activation was recently challenged (82).

The activation of Raf-1 is required for the subsequent multistep events to occur at the plasma membrane following the relief from autoinhibition. Agonists such as insulin and growth factors stimulate the phosphorylation of several residues, including serine-338, tyrosine-341, tyrosine-491, and serine-494 (83). Phosphorylation at serine-338 and tyrosine-341 is a critical step for Raf activation (83) and serine-338 phosphorylation appears to be a good qualitative indicator of Raf-1 activation.

MEK1 (MKK1) and MEK2 (MKK2) contain a proline-rich sequence necessary for the interaction of MEK with Raf-1 (84). MEKs are phosphorylated by Raf-1 on two serine residues (serine-217 and -221), which are necessary for full activation. MEK1 and MEK2 activate ERK1 (p44 MAPK) and ERK2 (p42 MAPK) via phosphorylation of a threonine–glutamate–tyrosine motif in the activation loop. ERK is a proline-directed serine/threonine kinase at the end of this pathway with more than 50 identified substrates, including transcription factors, MAPK-activated protein kinase-2, and the p90 ribosomal S6 kinase (85). ERK activation has traditionally been associated primarily with cell proliferation.

The stress-activated protein kinases (SAPKs) such as JNK, p38 MAPK, and ERK5 are slightly activated by insulin or growth factors but vigorously activated by stress signals (UV irradiation, heat or cold shock, osmotic stress, mechanical shear stress, oxidative stress), cytokines, and G protein–coupled receptor agonists (86). The SAPKs are involved in the regulation of growth arrest, apoptosis, and proliferation. The SAPKs are activated through a similar kinase cascade as ERK, although some different mechanisms have been noted. MKK4/SEK1 and MKK7 phosphorylate and activate JNK, whereas p38 MAPK is activated by MKK3 and MKK6. At the level of the MKKK, many kinases activating either or both JNK and p38 MAPK have been identified by overex-pression or dominant-negative experiments (87).

2.5 Crosstalk Between PI3K and MAPKs

A number of studies have suggested that the MAPK and the PI3K pathways cross-talk on several levels (88, 89, 90, 91, 92, 93, 94, 95, 96). But the interrelationship between MAPK and PI3K signaling pathways has been controversial. This may reflect differences associated with cell context and activators of the signaling cascades.

Studies of the relevance of PI3K signaling for the activation of ERK are inconclusive, with both increased and decreased ERK activation being reported. Although ERK1/2 activation may occur independently of PI3K (97, 98, 99), inhibitors of PI3K have been reported to inhibit insulin- and growth factor-induced increases in ERK1/2 activity in a number of cell types such as the rat skeletal muscle cell line L6, rat adipocytes, and hepatic stellate cells (100, 101, 102). In contrast, Akt phosphorylates serine-259, located in the regulatory domain of Raf-1, resulting in the inactivation of Raf-1 (103,104). Moelling et al. (92) showed that the PI3K/Akt pathway inhibited the Ras/Raf-1/MEK/ERK pathway at the level of Raf-1 and Akt. Thus, the PI3K-dependent signaling pathway can either stimulate or inhibit ERK activation and this likely depends on the cell context and the type of stimuli, as well as the concentration and period of treatment (88,92).

JNK activity is elevated in obesity and an absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and enhanced insulin receptor signaling capacity in two different models of mouse obesity (105). Lee et al. (96) showed that insulin-stimulated JNK associated with IRS1 and phosphorylated IRS1 at serine-307 in mouse embryo fibroblasts and 3T3-L1 adipocytes, and that this interaction inhibited insulin signaling. These results suggest that prolonged activation of JNK inhibits IRS-associated PI3K activity and can be a crucial mediator of insulin resistance.

2.6 Phosphatases

The phosphorylation of tyrosine residues in proteins by kinases plays a key role in the regulation of cell signaling and gene expression. The level of tyrosine phosphorylation in receptors and their downstream substrates is dynamically and precisely regulated by two types of enzymes, protein tyrosine kinases, which catalyze the phosphorylation of tyrosine residues, and protein tyrosine phosphatases (PTPs), which dephosphorylate the phosphotyrosine residues (106,107). Disregulated PTP activity may lead to aberrant tyrosine phosphorylation, which may contribute to disease, including cancer and diabetes (108,109). PTPs can be divided into tyrosine-specific and dual-specific subfamilies, based on their substrate specificity. The dual-specificity subfamily recognizes phosphotyrosine, phosphothreonine and phosphoserine residues. Tyrosine-specific PTPs can be further classified as intracellular and receptor-like PTPs. Intracellular PTPs possess a single conserved catalytic domain, and the N- or C-terminus that appears to play a regulatory or targeting role. PTP-1B and SHP-2, an SH2-containing PTP-2, belong to this subfamily and are key regulators that control the intracellular phosphotyrosine level. Receptor-like PTPs, exemplified by CD45 and PTPα, contain one or two cytoplasmic catalytic domains, a single transmembrane region and an extracellular domain. The extracellular domains have structures found in cell-adhesion molecules, suggesting a role for this subfamily of PTPs in cell–cell and/or cell–extracellular matrix interactions. Dual-specificity PTPs include the MAPK phosphatases (MKPs) and cell cycle regulator Cdc25 phosphatases.

PTP-1B, the first mammalian PTP to be purified to homogeneity (110), is widely expressed and localized predominantly to the endoplasmic reticulum through a cleavable proline-rich C-terminal segment (111). Cleavage of the C-terminal 35 amino acids of PTP-1B appears to release this phosphatase from the endoplasmic reticulum and increase its specific activity (112). PTP-1B deficiency in mice results in enhanced insulin sensitivity, as demonstrated by an increased insulin-stimulated phosphorylation of the insulin receptor in muscle and liver, an improved glucose clearance in glucose and insulin tolerance tests, and a significant reduction in fed glucose levels (113). This study suggests that PTP-1B is a negative regulator of the insulin-stimulated signal transduction pathway by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase (114). The association between insulin receptor and PTP-1B has been demonstrated using the substrate trapping method, immunoprecipitation, and immunoblot analysis (115,116). The mechanism(s) for regulation of PTP-1B activity is unclear. Recently, it has been reported that insulin-stimulated intracellular hydrogen peroxide production may reversibly oxidize PTP-1B, resulting in inhibition of PTP-1B and enhancement of the insulin signaling cascade (117,118). Several groups have reported that phosphorylation of PTP-1B by the insulin receptor and other protein kinases affects PTP-1B enzyme activity (119, 120, 121). In addition to the insulin receptor, PTP-1B has other targets such as EGF receptor and Src kinase (122,123).

The SHP-2 phosphatase contains two tandem SH2 domains that mediate the binding of SHP-2 to phosphorylated tyrosine residues on other molecules. In the resting state, SHP-2 activity is repressed, but its mechanism of activation is unclear. SHP-2 plays a positive and/or negative role in transducing signals relayed from RTKs. For example, introduction of the catalytically inert SHP-2 markedly inhibited activation of ERK in response to EGF and insulin stimulation (124,125). Chen et al. (126) also reported that overexpression of dominant-negative SHP-2 resulted in a modest impairment of insulin-stimulated glucose transporter 4 translocation, suggesting SHP-2 may play a minor role as a positive modulator of the metabolic effects of insulin. In contrast, Ouwens et al. (127) reported that expression of SHP-2 in cells resulted in a negative regulation of IRS-1 phosphorylation, PI3K activation, and stimulation of glycogen synthesis in response to insulin. Thus, it remains to be seen whether SHP-2 plays a major physiological role in insulin signaling.

The dual specificity MKPs are able to dephosphorylate both phosphotyrosine and phosphothreonine residues in the activation loop of MAPKs and inactivate them. In mammalian cells, at least 10 MKPs have been identified and individual MKPs display differential selectivity toward different MAPK family members and MKPs are localized to the nucleus or cytoplasm (128, 129, 130).

In general, serine/threonine protein phosphatases can be classified into the phosphoprotein phosphatase (PPP) and Mg2+-dependent protein phosphatase (PPM) gene families on the basis of similarity in the primary amino acid sequence between the different catalytic subunits (131). The PPP family includes the most abundant protein phosphatases—PP1, PP2A, and PP2B (calcineurin)—as well as more recently cloned enzymes such as PP4 (also known as PPX), PP5, PP6, and PP7. Five PPC2 isoforms, together with the pyruvate dehydro-genase phosphatase, constitute the gene family PPM. The catalytic subunit of PP1 has four mammalian isoforms and this phosphatase is inhibited by the cell-permeable toxins okadaic acid and calyculin A and the membrane-impermeable agent microcystin (132,133). PP2A is spontaneously active and inhibited by the inhibitors of PP1 whereas PP2B, a Ca2+-dependent protein, is not inhibited by these inhibitors (134). In response to insulin, PP1 is activated and catalyzes the insulin-mediated dephosphorylation of metabolic enzymes. The glycogen-associated form of PP1 dephosphorylates both glycogen synthase (resulting in enzyme activation) and glycogen phosphorylase (resulting in inactivation) to provide insulin-mediated coordination of glycogen metabolism (135). PP2A contributes to the dephosphorylation and regulation of MAPKs (81,136).

As discussed in Subheading 2.3., many different phosphorylated derivatives of PI play diverse roles in cellular signaling. The versatility of these molecules as cellular signals results from PH domain specificity in recognizing particular configurations of the inositol phosphate headgroup. Much attention has focused on PI(3,4,5)P3 as an intracellular second messenger produced rapidly via the action of PI3K in response to many divergent cellular stimuli. Phosphatase and tensin homologue deleted on chromosone 10 (PTEN) was discovered in 1997 as a new tumor suppressor and serves as an unusual phosphatase whose primary target is PI(3,4,5)P3. The 3-phosphorylated inositol lipids, PI(3,4,5)P3 and PI(3,4)P2, are the most efficient substrates for PTEN, which removes phosphate from the D-3 position of the inositol ring.

Evidence suggests that protein stability, localization, and transcription of the PTEN gene regulate the function of PTEN. The regulation of stability and localization appears to be achieved through the C-terminal tail of PTEN via phosphorylation of multiple serine and threonine residues. Recently, it has been shown that PI3K activation stimulates PTEN phosphorylation, suggesting that one of the kinases activated by PI3K is likely to be involved in PTEN phosphorylation (137).

3 Methods for Determination of Signalting Pathways Involved in the Regulation of Drug Metabolizing Enzyme Gene and Protein Expression

3.1 Methods for Examination of Protein Kinase Activity and Phosphorylation

Phosphorylation plays an essential role in the regulation of most protein kinases. Phosphorylation of specific residues is a major determinant of protein kinase activity. For example, the activation of Akt by insulin or growth factors is accompanied by phosphorylation on threonine-308 in the kinase domain and serine-473 in the C-terminal regulatory domain. Activation of Akt and phosphorylation of both these residues are abolished by treatment with the PI3K inhibitors, wortmannin or LY294002, prior to stimulation with an agonist such as insulin.

3.1.1 Immunoblot Analysis

Since first reported by Ross et al. (138), antibodies reactive with phosphoresidues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine) have become invaluable tools for isolating phosphorylated proteins and examining phosphorylation states. Phospho-specific antibodies for many protein kinases have been developed and these antibodies can be used for immunoblot analysis, immunoprecipitation, immunocytochemistry, and flow cytometry. In general, immunoblot analysis, coimmunoprecipitation, and kinase activity assays are the most frequently used methods for examination of protein kinase activation. Phospho-specific antibodies against a number of kinases and receptors are commercially available, and are used in standard immunoblotting procedures. If phospho-specific antibodies are not available, the kinase or receptor can be immunoprecipitated followed by immunoblotting with antiphosphotyrosine/serine/threonine antibodies.

Protein kinase activation usually occurs within a few minutes of agonist binding to a receptor (e.g., insulin and growth factors), suggesting that the phosphorylation state is dynamically regulated. Thus, inhibition of phosphatase activity is very important during preparation of cell lysates. Treatment of cells with phosphatase inhibitors may result in activation of kinases. Generally, cell lysis buffer contains phosphatase inhibitors such as sodium ortho-vanadate, sodium fluoride, ethylenebis(oxyethylenenitrilo)tetraacetic acid, and okadaic acid, to prevent dephosphorylation of protein kinases and other phosphoproteins. In immunoblot analysis, Laemmli sample buffer that contains sodium dodecyl sulfate and dithiothreitol can be used directly for making cell lysates.

3.1.2 Kinase Activity Assays

For protein kinase assays, phospho-specific antibodies to protein kinases have been used for selectively immunoprecipitating activated protein kinases from cell lysates. This method depends on the availability of a specific immunopre-cipitating antibody that does not interfere with the kinase activity, but many of these are commercially available. Protein kinase activity can be assayed by incorporation of phosphate from ATP into a synthetic peptide substrate based on the sequences of the phosphorylation sites on the target substrate protein. Many protein kinases phosphorylate the short peptide substrate with kinetic parameters similar to those of the native target proteins. In some cases, however, protein kinases that recognize or require an aspect of 3-D structure for their target, in addition to the primary sequence, will phosphorylate synthetic peptides poorly or not at all. Kinases that fall into this class must be assayed using the native protein target as substrate, or at least an expressed domain of the substrate that contains the requisite recognition features. Most protein kinase assays using synthetic peptides use radioactive ATP, resulting in a radiolabeled phosphopeptide that can be quantified by scintillation counting. Recently, nonradioactive kinase assays employing phospho-specific antibodies to the substrate protein have been developed and allow detection and quantification of kinase activity following immunoprecipitation of an active kinase.

3.2 Chemical Inhibitors of Protein Kinases

A widely used approach for examining the role of a kinase or kinase family in a cell signaling pathway is the pharmacological inhibition of the kinase (Fig. 5). To elucidate the signaling function of individual protein kinases and phosphatases, inhibitors should be potent, highly specific, and cell-membrane permeable. Many inhibitors of protein kinases and phosphatases have been developed as therapy for diseases such as cancer, inflammation, and diabetes. The vast majority of protein kinase inhibitors have been designed to target the ATP-binding site of protein kinases (139). Expectations for inhibitor specificity were initially poor because the number of protein kinases encoded in the human genome is estimated to be in excess of 500, and the significant number of other cellular enzymes that use ATP further complicates the issue. Nevertheless, several ATP-binding site-directed protein kinase inhibitors have been developed with a high degree of selectivity. Based on the numerous structures of complexes with ATP, it is clear that the ATP-binding cleft has regions that are not occupied by ATP and these regions show structural diversity among kinases (140). In general, these inhibitors are added to cells prior to agonist addition. For longer periods of treatment (e.g., 24–48 h), the inhibitor may need to be replenished because the inhibitor half-life may be limited.
Fig. 5.

Kinase inhibitors acting on insulin signaling pathways.

The inhibitors PD98059 and U0126 bind to the inactive form of MEK, preventing its activation by Raf-1 and other upstream activators (141,142). These inhibitors do not compete with ATP and do not inhibit the phosphorylation of MEK, and thus are likely to have a distinct binding site on MEK. Quantitative evaluation of the steady-state kinetics of MEK inhibition by these compounds shows that U0126 has higher affinity than PD98059 (142). In a comparison of multiple kinase inhibitors, the MEK1 and MEK2 inhibitors appeared to be the most specific kinase inhibitors tested (143). But both of these inhibitors have recently been shown to inhibit activation of the ERK5 pathway through direct effects on MEK5 (144). It is recommended that PD98059 or U0126 be added to cells at a concentration of 50–100 μM or 5–25 μM, respectively.

SB203580 and SB202190, a class of pyridinyl imidazoles, are relatively specific inhibitors of p38 MAPK α and β, but not p38 MAPK γ and δ, at a concentration of 10 μM (145,146). However, these inhibitors were reported to inhibit the activation of PDK1 and its downstream effectors, including Akt and p70 S6 kinase (147,148), although PDK1 activity remained unaffected by in vitro incubation with SB203580 or SB202190 (143). We have found that in primary cultured rat hepatocytes, these p38 MAPK inhibitors failed to affect insulin-mediated Akt phosphorylation (40). These compounds bind the ATP-binding cleft of the low-activity p38 MAPK, which binds ATP poorly (149). As a consequence of binding the unphosphorylated form, these inhibitors appear to interfere with the activation of p38 MAPK. Generally, SB203580 and SB202190 completely inhibit p38 MAPK at a concentration of 10 μM.

SP600125, an anthrapyrazolone inhibitor of JNK1, JNK2 and JNK3, has been reported to inhibit JNKs through a reversible ATP-competition (150). A number of studies have reported that the compound prevents the expression of several anti-inflammatory genes in cell-based assays and the activation of AP1 in synoviocytes (150,151). The inhibitor is starting to be used more widely as a JNK inhibitor. However, Bain et al. (152) recently reported that SP600125 was a relatively weak inhibitor of JNK isoforms and also inhibited other protein kinases with similar or greater potency. Care must be used, therefore, when employing this inhibitor and in the interpretation of resulting data. For inhibition of JNKs, SP600126 has been used at a concentration of 10–25 μM.

SU6656 (153) and the related pyrazolopyrimidine, PP1 (154), were developed as inhibitors of the Src family of enzymes. PP1 was originally described as a selective, ATP-competitive inhibitor of Src family kinases and has been widely used to investigate the contribution of Src kinases to a number of biological functions (154). It is recommended that SU6656 be added to cells at a concentration of 1–5 μM.

Rapamycin, a potent immunosuppressant, rapidly inactivates p70 S6 kinase and prevents the activation of this kinase by all known agonists (155,156). Rapamycin binds to the immunophilin FK506 binding protein 12, and the resultant complex interacts with the protein kinase mTOR/FKBP 12-rapamycin-associated protein, thereby inhibiting it. This leads to the dephosphorylation and inactivation of p70 S6 kinase (157). Generally, rapamycin completely inhibits p70 S6 kinase at a concentration of 100 nM.

GF109203X (bisindolylmaleimide I; Gö6850) and Ro-31-8220 are bisin-dolylmaleimides that differ from each other in two functional groups and are analogues of staurosporine (158,159). These inhibitors, which compete for the ATP binding site on PKC, have approx 100-fold selectivity for PKC over PKA (160). They are both potent inhibitors of the α, β, and γ isoforms of PKC with IC50 values in the nanomolar range in vitro. However, micromolar concentrations of GF109203X are required to inhibit atypical PKCs (161). These classes of compounds may also have the ability to selectivity inhibit PKC isoforms. Gö6976, another staurosporine-related compound, inhibits α- and β1-PKCs when utilized at nanomolar concentrations, but fails to inhibit δ-, ε-, and ζ-PKC isoforms (161). It is recommended that GF109203X be added to cells at a concentration of 1–10 μM.

Wortmannin and LY294002 are cell-permeable inhibitors of PI3K (162,163). Wortmannin, an irreversible inhibitor, alkylates a lysine residue at the putative ATP binding site of p110α of PI3K and LY294002 is a pure competitive inhibitor of ATP. It is recommended that wortmannin or LY294002 be added to cells at a concentration of 100–500 nM or 10–20 μM, respectively. At higher concentrations, wortmannin inhibits a number of other kinases, including the class 2 PI3K (164,165). If a longer incubation time is required, LY294002 is the inhibitor of choice rather than wortmannin, because of its higher stability in aqueous solution.

For longer treatment durations in highly metabolically competent cells, such as primary hepatocytes, the concentrations of protein kinase inhibitor recommended earlier may not be sufficient to inhibit each target protein kinase activity. Thus, higher concentrations of most of these inhibitors are often required to offset metabolism of the inhibitor, and care must therefore be exercised in the interpretation of these data.

In primary cultured rat hepatocytes, wortmannin and LY294002 effectively inhibit both basal and insulin-mediated Akt phosphorylation (Fig. 6 and 7). We have used these inhibitors to demonstrate that PI3K plays an obligatory role in the insulin-mediated induction of mEH protein (Fig. 8) and the insulin-mediated suppression of CYP2E1 mRNA expression (37,48).
Fig. 6.

The effects of the PI3K inhibitors, wortmannin (A) or LY294002 (B), on phosphorylation of Akt in rat hepatocytes cultured in the absence of insulin. Hepatocytes were treated with wortmannin or LY294002 for 4.5 h. Untreated (UT) hepatocytes were cultured in the absence of insulin and inhibitor. Phospho-Akt levels were normalized to total Akt levels. Values are shown as a percentage of the level of phospho-Akt/total-Akt in untreated hepatocytes (100%=1245 arbitrary densitometry units of phospho-Akt and 1522 arbitrary densitometry units of Akt [A], 705 arbitrary densitometry units of phospho-Akt and 1471 arbitrary densitometry units of Akt [B]). Data are means ± SD of Western blot band densities of two preparations of cell lysates from a single hepatocyte preparation. (Reprinted from ref. 40 with permission.)

Fig. 7.

The effects of the PI3K inhibitors, wortmannin (A) or LY294002 (B), on the insulin-mediated phosphorylation of Akt in primary cultured rat hepatocytes. Hepatocytes were treated with wortmannin or LY294002 for 1.5 h before addition of 10 nM insulin for 3 h. Untreated (UT) hepatocytes were cultured in the absence of insulin and inhibitor. Phospho-Akt levels were normalized to total Akt levels. Values are shown as a percentage of the level of phospho-Akt/total-Akt in untreated hepatocytes (100%=84 arbitrary densitometry units of phospho-Akt and 776 arbitrary densitometry units of Akt [A], 36 arbitrary densitometry units of phospho-Akt and 473 arbitrary densitometry units of Akt [B]). Data are means ± SD of Western blot band densities of two preparations of cell lysates from a single hepatocyte preparation. (Reprinted from ref. 40 with permission.)

Fig. 8.

Immunoblot analysis of the effects of the PI3K inhibitors, wortmannin (A) or LY294002 (B), on the insulin-mediated increase in mEH protein levels in primary cultured rat hepatocytes. (A) Hepatocytes were treated with wortmannin alone, or for 1.5 h prior to addition of 10 nM insulin for 24 h. (B) Hepatocytes were treated with LY294002 alone, or 1.5 h prior to addition of 10 nM insulin for 24 h. Untreated (UT) hepatocytes were cultured in the absence of insulin and inhibitors. Values are shown as a percentage of the level monitored in untreated hepatocytes (100%=413 arbitrary densitometry units

3.3 Dominant-Negative Protein Kinase Constructs

The activity of a protein kinase can be interfered with by expression of a dominant-negative mutant. The generation of dominant-negative mutants involves the design of an inactive form of the protein that can sequester interacting proteins. Some knowledge of the mechanism of regulation or function of the protein of interest is helpful when designing these molecules. In general, the activity of protein kinases requires the phosphorylation of specific residues for activation and the binding of ATP to a conserved protein motif for phosphorylation of effector proteins. Thus, the point mutation of the phosphorylation site or ATP-binding region can produce an inactivated kinase or kinase-dead mutant, respectively (166,167). Overexpression of an inactive form of the kinase may act as a dominant-negative by sequestering interacting proteins or cofactors and thus inhibiting the activity of the endogenous wild-type kinase. Many protein kinases are inactive in resting cells and this basal inhibition is achieved by interaction with a regulatory protein or an inhibitory domain within the same polypeptide. Thus, in some cases, overexpression of a regulatory protein or an inhibitory domain can reduce or inhibit the ability of the pathway to stimulate the endogenous protein. Similarly, overexpression of a pseudosubstrate domain that can bind the enzyme but cannot be converted to product can often result in inhibition of signaling, as it will compete with the endogenous substrate (168).

DNA constructs encoding inactive kinase mutants must be transported through the cell membrane and into the nucleus, to inhibit signaling pathways through their expression. There are several well-established techniques that allow transient transfection of recombinant DNA into cells in culture. These methods generally involve the permeabilization of cell membranes by chemical or electrical means, or the use of viral constructs that can recognize specific receptors on the cell surface, resulting in cellular uptake. A variety of viral systems, including adenoviruses and retroviruses, have become available for transporting recombinant DNA into cells (169). The DNA can either be incorporated into the viral genome or be chemically linked to the exterior of the virion. After transfection of adenovirus into a mammalian cell, viral production may be monitored with green fluorescent protein (GFP), which is encoded by a gene incorporated into the viral backbone (170). The most common methodologies have been reviewed in detail elsewhere (170,171).

3.4 siRNA

In 1998, Fire and Mello described a new technology that was based on the silencing of specific genes by double-stranded RNA (dsRNA) and termed RNA interference (RNAi) (172). RNAi consists of the presentation of a “triggering” dsRNA that is subsequently processed into 21–25 base-pair small interfering RNAs (siRNAs) through the action of the Dicer enzyme (RNase III endonu-clease) (173, 174, 175). siRNAs with 2-nucleotide 3′-end overhangs are then incorporated into a multisubunit RNA-induced silencing complex, which targets their complementary RNA transcript for enzymatic degradation (176). The siRNA-induced degradation of mRNA is highly sequence-specific, to the extent that even a one- or two-nucleotide difference in the targeting recognition sequence hampers RNAi function.

In contrast to siRNAs, small temporal RNA (stRNA) molecules, which represent a large group of small transcripts called micro-RNAs, mediate gene suppression by inhibiting translation of target mRNA (177,178). In common with siRNAs, Dicer is also involved in the processing of the 21- to 23-nucleotide stRNAs from approx 70-nucleotide stable hairpin precursors (179). But stRNAs are stem-loops that are processed into an imperfect complementary dsRNA that inhibit protein translation of an imperfectly matched target sequence, which is almost invariably located at the 3′-untranslated region of the target mRNA (180).

In mammalian somatic cells, dsRNAs longer than 30 nucleotides activate an antiviral defense mechanism that includes the production of interferon and activation of dsRNA-dependent protein kinase, resulting in inhibition of protein synthesis initiation and stimulation of apoptosis (181,182). One mechanism for dealing with these nonspecific dsRNA responses is to create dsRNA triggers of fewer than 30 base pairs in length. Both siRNA and stRNA are long enough to induce sequence-specific suppression, but short enough to evade the host defense response. Although the use of siRNAs to silence genes in vertebrate cells was reported only a few years ago, the emerging literature indicates that most vertebrate genes can be studied with this technology.

Several laboratories demonstrated that synthesized dsRNAs induced sequence-specific gene silencing when transiently transfected into mammalian cells (183,184). Factors that could ultimately limit the usefulness of siRNAs include a relatively short and transient period of activity. The longevity of silencing is dependent on abundance of mRNA and protein, stability of protein, the half-life of the silencing complex, and cell division rate. Generally the siRNA directs rapid reduction in mRNA levels that is readily observed in 18 h or less and siRNA-mediated RNAi lasts for 3–5 d for most cell lines (185).

Recently a number of studies have reported the success of using RNA polymerase III promoters, such as U6 or H1, to direct in vivo synthesis of functional siRNAs (186, 187, 188, 189, 190, 191). These siRNAs have been expressed in two ways. In the first case, hairpin constructs are expressed from a single RNA polymerase III promoter. The resulting RNAs are predicted to form hairpins containing 19- to 29-nucleotide stems that match target sequences precisely, three- or nine-nucleotide loops and 3′ overhangs of four or fewer uridines. It is believed that these hairpin RNAs are processed by Dicer to active siRNAs in vivo (192). In the second case, coding and noncoding strands of a potential siRNA are driven from separate promoters and the expressed transcripts anneal in the cell nucleus. The hairpin siRNA strategy appears to inhibit gene expression more efficiently than the duplex siRNAs expressed from two separate plasmids (192). The use of a plasmid-based RNA polymerase III promoter system to intracellularly produce siRNAs could allow for a longer period of expression as compared with exogenously added siRNAs.

An alternative approach to prolong siRNA-mediated inhibition of gene expression is the introduction of modified nucleotides into chemically synthesized RNA. Amarzguioui et al. (193) reported that siRNA generally tolerated mutations in the 5′-end, while the 3′-end exhibited low tolerance. An siRNA with two 2′-O-methyl RNA nucleotides at the 5′-end and four methylated monomers at the 3′-end was as active as its unmodified counterpart and led to a prolonged silencing effect in cell culture (193).

The effectiveness of an siRNA is likely to be determined by the accessibility of its target sequence in the intended substrate. It has been suggested that the first 50–100 nucleotides of an mRNA sequence, downstream of the translation initiation sequence, should be used to target a gene and that 5′- or 3′-untranslated regions, as well as highly conserved domains (i.e., catalytic, ligand binding, etc.), should be avoided, as they are likely to contain regulatory protein binding sites (190). However, successful gene inhibition has been reported for siRNAs targeting various sequences, including the 3′-untranslated region (194). There are no reliable ways to predict or identify the “ideal” sequence for an siRNA. However, targeting different regions of a given mRNA might give different results (185). Generally, siRNAs become susceptible to RNase H; therefore, the degree of the RNase H sensitivity of a given probe reflects the RNase H accessibility of the chosen sites. In practical terms, it might be just as easy to construct and test several siRNAs.

4 Conclusion

It is becoming increasingly clear that endogenous factors, including hormones and growth factors, play an important role in the regulation of drug-metabolizing enzyme expression in both physiological and pathophysiological conditions. Our laboratory has used phospho-specific antibodies and chemical inhibitors of protein kinases to define the signaling pathways involved in insulin- and glucagon-mediated regulation of several drug-metabolizing enzymes (40,48). Small molecule chemical inhibitors of protein kinases used for this purpose in many studies, however, have been reported to have specificity problems, although many chemicals have been considered to be reasonably selective inhibitors for each target protein kinase. As with all pharmacological tools, interpretation of experiments with these protein kinase inhibitors requires caution. It is advisable to conduct experiments with at least two pharmacologically distinct inhibitors wherever possible. Dominant-negative kinase constructs allow for more kinase-specific inhibition. Recently, RNAi methods have opened new opportunities for investigators to study cell signaling pathways by leading to a highly specific mRNA degradation. Furthermore, retrovirus or adenovirus vectors have been developed for use in carrying dominant-negative kinase constructs or siRNA-expressing DNA templates into cells to mediate gene-specific silencing in cells or animals. Thus, RNAi using siRNAs to silence specific genes is a very promising method for determination of cell signaling pathways involved in protein expression in response to hormones and growth factors.


  1. 1.
    Duvaldestin P, Mahu J-L, Berthelot P. Effect of fasting on substrate specificity of rat liver UDP-glucuronosyltransferase. Biochim Biophys Acta 1975;384:81–86.PubMedGoogle Scholar
  2. 2.
    Abernethy DR, Greenblatt DJ, Divoll M, Shader RI. Enhanced glucuronide conjugation of drugs in obesity: studies of lorazepam, oxazepam, and acetaminophen. J Lab Clin Med 1983;101:873–880.PubMedGoogle Scholar
  3. 3.
    Hong JY, Pan JM, Gonzalez FJ, Gelboin HV, Yang CS. The induction of a specific form of cytochrome P-450 (P-450j) by fasting. Biochem Biophys Res Commun 1987;142:1077–1083.PubMedCrossRefGoogle Scholar
  4. 4.
    Bellward GD, Chang T, Rodrigues B, et al. Hepatic cytochrome P-450j induction in the spontaneously diabetic BB rat. Mol Pharmacol 1988;33:140–143.PubMedGoogle Scholar
  5. 5.
    Thomas H, Schladt L, Knehr M, Oesch F. Effect of diabetes and starvation on the activity of rat liver epoxide hydrolases, glutathione S-transferases and peroxisomal beta-oxidation. Biochem Pharmacol 1989;38:4291–4297.PubMedCrossRefGoogle Scholar
  6. 6.
    Yamazoe Y, Murayama N, Shimada M, Yamauchi K, Kato R. Cytochrome P450 in livers of diabetic rats: regulation by growth hormone and insulin. Arch Biochem Biophys 1989;268:567–575.PubMedCrossRefGoogle Scholar
  7. 7.
    Barnett CR, Gibson GG, Wolf CR, Flatt PR, Ioannides C. Induction of cytochrome P450III and P450IV family proteins in streptozotocin-induced diabetes. Biochem J 1990;268:765–769.PubMedGoogle Scholar
  8. 8.
    Song BJ, Veech RL, Saenger P. Cytochrome P450IIE1 is elevated in lymphocytes from poorly controlled insulin-dependent diabetics. J Clin Endocrinol Metab 1990; 71:1036–1040.PubMedCrossRefGoogle Scholar
  9. 9.
    Donahue BS, Skottner-Lundin A, Morgan ET. Growth hormone-dependent and-independent regulation of cytochrome P-450 isozyme expression in streptozotocin-diabetic rats. Endocrinology 1991;128:2065–2076.PubMedCrossRefGoogle Scholar
  10. 10.
    Chaudhary IP, Tuntaterdtum S, McNamara PJ, Robertson LW, Blouin RA. Effect of genetic obesity and phenobarbital treatment on the hepatic conjugation pathways. J Pharmacol Exp Ther 1993;265:1333–1338.PubMedGoogle Scholar
  11. 11.
    Ronis MJ, Huang J, Crouch J, et al. Cytochrome P450 CYP 2E1 induction during chronic alcohol exposure occurs by a two-step mechanism associated with blood alcohol concentrations in rats. J Pharmacol Exp Ther 1993;264:944–950.PubMedGoogle Scholar
  12. 12.
    Shimojo N, Ishizaki T, Imaoka S, Funae Y, Fujii S, Okuda K. Changes in amounts of cytochrome P450 isozymes and levels of catalytic activities in hepatic and renal microsomes of rats with streptozocin-induced diabetes. Biochem Pharmacol 1993; 46:621–627.PubMedCrossRefGoogle Scholar
  13. 13.
    Van de Wiel JA, Fijneman PH, Teeuw KB, Van Ommen B, Noordhoek J, Bos RP. Influence of long-term ethanol treatment on rat liver biotransformation enzymes. Alcohol 1993;10:397–402.PubMedCrossRefGoogle Scholar
  14. 14.
    Runge-Morris M, Vento C. Effects of streptozotocin-induced diabetes on rat liver sulfotransferase gene expression. Drug Metab Dispos 1995;23:455–459.PubMedGoogle Scholar
  15. 15.
    Visser TJ, van Haasteren GA, Linkels E, Kaptein E, van Toor H, de Greef WJ. Gender-specific changes in thyroid hormone-glucuronidating enzymes in rat liver during short-term fasting and long-term food restriction. Eur J Endocrinol 1996; 135:489–497.PubMedCrossRefGoogle Scholar
  16. 16.
    Braun L, Coffey MJ, Puskas F, et al. Molecular basis of bilirubin UDP-glucuronosyltransferase induction in spontaneously diabetic rats, acetone-treated rats and starved rats. Biochem J 1998;336:587–592.PubMedGoogle Scholar
  17. 17.
    Kardon T, Coffey MJ, Banhegyi G, et al. Transcriptional induction of bilirubin UDP-glucuronosyltransrase by ethanol in rat liver. Alcohol 2000;21:251–257.PubMedCrossRefGoogle Scholar
  18. 18.
    Agius C, Gidari AS. Effect of streptozotocin on the glutathione S-transferases of mouse liver cytosol. Biochem Pharmacol 1985;34:811–819.PubMedCrossRefGoogle Scholar
  19. 19.
    Grant MH, Duthie SJ. Conjugation reactions in hepatocytes isolated from streptozotocin-induced diabetic rats. Biochem Pharmacol 1987;36:3647–3655.PubMedCrossRefGoogle Scholar
  20. 20.
    Mukherjee B, Mukherjee JR, Chatterjee M. Lipid peroxidation, glutathione levels and changes in glutathione-related enzyme activities in streptozotocin-induced diabetic rats. Immunol Cell Biol 1994;72:109–114.PubMedCrossRefGoogle Scholar
  21. 21.
    Raza H, Ahmed I, Lakhani MS, Sharma AK, Pallot D, Montague W. Effect of bitter melon (Momordica charantia) fruit juice on the hepatic cytochrome P450-dependent monooxygenases and glutathione S-transferases in streptozotocin-induced diabetic rats. Biochem Pharmacol 1996;52:1639–1642.PubMedCrossRefGoogle Scholar
  22. 22.
    Wasserman WW, Fahl WE. Functional antioxidant responsive elements. Proc Natl Acad Sci USA 1997;94:5361–5366.PubMedCrossRefGoogle Scholar
  23. 23.
    Kang KW, Cho MK, Lee CH, Kim SG. Activation of phosphatidylinositol 3-kinase and Akt by tert-butylhydroquinone is responsible for antioxidant response element-mediated rGSTA2 induction in H4IIE cells. Mol Pharmacol 2001;59:1147–1156.PubMedGoogle Scholar
  24. 24.
    Dong ZG, Hong JY, Ma QA, et al. Mechanism of induction of cytochrome P-450ac (P-450j) in chemically induced and spontaneously diabetic rats. Arch Biochem Biophys 1988;263:29–35.PubMedCrossRefGoogle Scholar
  25. 25.
    Favreau LV, Schenkman JB. Composition changes in hepatic microsomal cytochrome P-450 during onset of streptozocin-induced diabetes and during insulin treatment. Diabetes 1988;37:577–584.PubMedCrossRefGoogle Scholar
  26. 26.
    Yamazoe Y, Murayama N, Shimada M, Imaoka S, Funae Y, Kato R. Suppression of hepatic levels of an ethanol-inducible P-450DM/j by growth hormone: relationship between the increased level of P-450DM/j and depletion of growth hormone in diabetes. Mol Pharmacol 1989;36:716–722.PubMedGoogle Scholar
  27. 27.
    Donahue BS, Morgan ET. Effects of vanadate on hepatic cytochrome P-450 expression in streptozotocin-diabetic rats. Drug Metab Dispos 1990;18:519–526.PubMedGoogle Scholar
  28. 28.
    Thummel KE, Schenkman JB. Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat. Mol Pharmacol 1990;37:119–129.PubMedGoogle Scholar
  29. 29.
    Tunon MJ, Gonzalez P, Garcia-Pardo LA, Gonzalez J. Hepatic transport of bilirubin in rats with streptozotocin-induced diabetes. J Hepatol 1991;13:71–77.PubMedCrossRefGoogle Scholar
  30. 30.
    Constantopoulos A, Matsaniotis N. Augmentation of uridine diphosphate glucuronyltransferase activity in rat liver by adenosine 3′,5′-monophosphate. Gastroenterology 1978;75:486–491.PubMedGoogle Scholar
  31. 31.
    Ricci GL, Fevery J. Treatment of rats with glucagon, vasointestinal peptide or secretin has a different effect on bilirubin and p-nitrophenol UDP-glucuronyl-transferase. Biochem Pharmacol 1988;37:3526–3528.PubMedCrossRefGoogle Scholar
  32. 32.
    Carrillo MC, Monti JA, Favre C, Carnovale CE. Acute regulation of hepatic glutathione S-transferase by insulin and glucagon. Toxicol Lett 1995;76:105–111.PubMedCrossRefGoogle Scholar
  33. 33.
    Kim SK, Woodcroft KJ, Novak RF. Insulin and glucagon regulation of glutathione S-transferase expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther 2003;305:353–361.PubMedCrossRefGoogle Scholar
  34. 34.
    De Waziers I, Garlatti M, Bouguet J, Beaune PH, Barouki R. Insulin down-regulates cytochrome P450 2B and 2E expression at the post-transcriptional level in the rat hepatoma cell line. Mol Pharmacol 1995;47:474–479.PubMedGoogle Scholar
  35. 35.
    Woodcroft KJ, Novak RF. Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes. Chem Biol Interact 1997;107:75–91.PubMedCrossRefGoogle Scholar
  36. 36.
    Woodcroft KJ, Novak RF. Insulin differentially affects xenobiotic-enhanced, cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther 1999;289:1121–1127.PubMedGoogle Scholar
  37. 37.
    Woodcroft KJ, Novak RF. The role of phosphatidylinositol 3-kinase, Src kinase, and protein kinase A signaling pathways in insulin and glucagon regulation of CYP2E1 expression. Biochem Biophys Res Commun 1999;266:304–307.PubMedCrossRefGoogle Scholar
  38. 38.
    Iber H, Li-Masters T, Chen Q, Yu S, Morgan ET. Regulation of hepatic cytochrome P450 2C11 via cAMP: implications for down-regulation in diabetes, fasting, and inflammation. J Pharmacol Exp Ther 2001;297:174–180.PubMedGoogle Scholar
  39. 39.
    Viitala P, Posti K, Lindfors A, Pelkonen O, Raunio H. cAMP mediated upregulation of CYP2A5 in mouse hepatocytes. Biochem Biophys Res Commun 2001;280: 761–767.PubMedCrossRefGoogle Scholar
  40. 40.
    Kim SK, Woodcroft KJ, Kim SG, Novak RF. Insulin and glucagon signaling in regulation of microsomal epoxide hydrolase expression in primary cultured rat hepatocytes. Drug Metab Dispos 2003;31:1260–1268.PubMedCrossRefGoogle Scholar
  41. 41.
    Iber H, Morgan ET. Regulation of hepatic cytochrome P450 2C11 by transforming growth factor-beta, hepatocyte growth factor, and interleukin-11. Drug Metab Dispos 1998;26:1042–1044.PubMedGoogle Scholar
  42. 42.
    Donato MT, Gomez-Lechon MJ, Jover R, Nakamura T, Castell JV. Human hepatocyte growth factor down-regulates the expression of cytochrome P450 isozymes in human hepatocytes in primary culture. J Pharmacol Exp Ther 1998;284:760–767.PubMedGoogle Scholar
  43. 43.
    Ching KZ, Tenney KA, Chen J, Morgan ET. Suppression of constitutive cytochrome P450 gene expression by epidermal growth factor receptor ligands in cultured rat hepatocytes. Drug Metab Dispos 1996;24:542–546.PubMedGoogle Scholar
  44. 44.
    De Smet K, Loyer P, Gilot D, Vercruysse A, Rogiers V, Guguen-Guillouzo C. Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes. Biochem Pharmacol 2001;61:1293–1303.PubMedCrossRefGoogle Scholar
  45. 45.
    Garcia MC, Thangavel C, Shapiro BH. Epidermal growth factor regulation of female-dependent CYP2A1 and CYP2C12 in primary rat hepatocyte culture. Drug Metab Dispos 2001;29:111–120.PubMedGoogle Scholar
  46. 46.
    Matsumoto M, Imagawa M, Aoki Y. Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I. Biochem J 2000;349:225–230.PubMedCrossRefGoogle Scholar
  47. 47.
    Desmots F, Rissel M, Gilot D, et al. Pro-inflammatory cytokines tumor necrosis factor α and interleukin-6 and survival factor epidermal growth factor positively regulate the murine GSTA4 enzyme in hepatocytes. J Biol Chem 2002;277: 17892–17900.PubMedCrossRefGoogle Scholar
  48. 48.
    Woodcroft KJ, Hafner MS, Novak RF. Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression. Hepatology 2002;35: 263–273.PubMedCrossRefGoogle Scholar
  49. 49.
    Perz M, Torlinska T. Insulin receptor—structural and functional characteristics. Med Sci Monit 2001;7:169–177.PubMedGoogle Scholar
  50. 50.
    Sparrow LG, McKern NM, Gorman JJ, et al. The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain. J Biol Chem 1997;272: 29460–29467.PubMedCrossRefGoogle Scholar
  51. 51.
    Cheatham B, Kahn CR. Cysteine 647 in the insulin receptor is required for normal covalent interaction between α-and β-subunits and signal transduction. J Biol Chem 1992;267:7108–7115.PubMedGoogle Scholar
  52. 52.
    Kahn CR. Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 1994;43:1066–1084.PubMedCrossRefGoogle Scholar
  53. 53.
    White MF, Kahn CR. The insulin signaling system. J Biol Chem 1994;269:1–4.PubMedGoogle Scholar
  54. 54.
    White MF. IRS proteins and the common path to diabetes. Am J Physiol 2002; 283:E413–E422.Google Scholar
  55. 55.
    Kao AW, Waters SB, Okada S, Pessin JE. Insulin stimulates the phosphorylation of the 66-and 52-kilodalton Shc isoforms by distinct pathways. Endocrinology 1997; 138:2474–2480.PubMedCrossRefGoogle Scholar
  56. 56.
    Sasaoka T, Kobayashi M. The functional significance of Shc in insulin signaling as a substrate of the insulin receptor. Endocr J 2000;47:373–381.PubMedCrossRefGoogle Scholar
  57. 57.
    Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003;284:31–53.PubMedCrossRefGoogle Scholar
  58. 58.
    Whiteley B, Glaser L. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor. J Cell Biol 1986;103:1355–1362.PubMedCrossRefGoogle Scholar
  59. 59.
    Tice DA, Biscardi JS, Nickles AL, Parsons SJ. Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl Acad Sci USA 1999;96:1415–1420.PubMedCrossRefGoogle Scholar
  60. 60.
    Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem 1998;67:481–507.PubMedCrossRefGoogle Scholar
  61. 61.
    Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655–1657.PubMedCrossRefGoogle Scholar
  62. 62.
    Shields JM, Pruitt K, McFall A, Shaub A, Der CJ. Understanding Ras: ‘it ain’t over’ til it’s over’. Trends Cell Biol 2000;10:147–154.PubMedCrossRefGoogle Scholar
  63. 63.
    Alessi DR, Deak M, Casamayor A, et al. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 1997;7:776–789.PubMedCrossRefGoogle Scholar
  64. 64.
    Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 1998;281:2042–2045.PubMedCrossRefGoogle Scholar
  65. 65.
    Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 2000;346:561–576.PubMedCrossRefGoogle Scholar
  66. 66.
    Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999;68:965–1014.PubMedCrossRefGoogle Scholar
  67. 67.
    Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 1996;15:6541–6551.PubMedGoogle Scholar
  68. 68.
    Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G. Rapamycin suppresses 5’TOP mRNA translation through inhibition of p70s6k EMBO J 1997;16:3693–3704.PubMedCrossRefGoogle Scholar
  69. 69.
    Kawasome H, Papst P, Webb S, et al. Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity. Proc Natl Acad Sci USA 1998; 95:5033–5038.PubMedCrossRefGoogle Scholar
  70. 70.
    Kohn AD, Takeuchi F, Roth RA. Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem 1996;271: 21920–21926.PubMedCrossRefGoogle Scholar
  71. 71.
    Conus NM, Hemmings BA, Pearson RB. Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k. J Biol Chem 1998;273:4776–4782.PubMedCrossRefGoogle Scholar
  72. 72.
    Dufner A, Andjelkovic M, Burgering BM, Hemmings BA, Thomas G. Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation. Mol Cell Biol 1999;19:4525–4534.PubMedGoogle Scholar
  73. 73.
    Farese RV. Insulin-sensitive phospholipid signaling systems and glucose transport. Update II. Exp Biol Med (Maywood) 2001;226:283–295.Google Scholar
  74. 74.
    Standaert ML, Bandyopadhyay G, Perez L, et al. Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes. J Biol Chem 1999;274:25308–25316.PubMedCrossRefGoogle Scholar
  75. 75.
    Standaert ML, Bandyopadhyay G, Kanoh Y, Sajan MP, Farese RV. Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites. Biochemistry 2001;40:249–255.PubMedCrossRefGoogle Scholar
  76. 76.
    Mendez R, Kollmorgen G, White MF, Rhoads RE. Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin. Mol Cell Biol 1997;17: 5184–5192.PubMedGoogle Scholar
  77. 77.
    Romanelli A, Martin KA, Toker A, Blenis J. p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex. Mol Cell Biol 1999;19:2921–2928.PubMedGoogle Scholar
  78. 78.
    Khosravi-Far R, Campbell S, Rossman KL, Der CJ. Increasing complexity of Ras signal transduction: involvement of Rho family proteins. Adv Cancer Res 1998;72: 57–107.PubMedCrossRefGoogle Scholar
  79. 79.
    Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Signal 2003;15:463–469.PubMedCrossRefGoogle Scholar
  80. 80.
    Tzivion G, Luo Z, Avruch J. A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. Nature 1998;394:88–92.PubMedCrossRefGoogle Scholar
  81. 81.
    Kubicek M, Pacher M, Abraham D, Podar K, Eulitz M, Baccarini M. Dephos-phorylation of Ser-259 regulates Raf-1 membrane association. J Biol Chem 2002; 277:7913–7919.PubMedCrossRefGoogle Scholar
  82. 82.
    Light Y, Paterson H, Marais R. 14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity. Mol Cell Biol 2002;22:4984–4996.PubMedCrossRefGoogle Scholar
  83. 83.
    Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ, Marais R. Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 1999;18:2137–2148.PubMedCrossRefGoogle Scholar
  84. 84.
    Catling AD, Schaeffer HJ, Reuter CW, Reddy GR, Weber MJ. A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function. Mol Cell Biol 1995;15:5214–5225.PubMedGoogle Scholar
  85. 85.
    Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res 1998;74:49–139.PubMedCrossRefGoogle Scholar
  86. 86.
    Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81: 807–869.PubMedGoogle Scholar
  87. 87.
    Hagemann C, Blank JL. The ups and downs of MEK kinase interactions. Cell Signal 2001;13:863–875.PubMedCrossRefGoogle Scholar
  88. 88.
    Duckworth BC, Cantley LC. Conditional inhibition of the mitogen-activated protein kinase cascade by wortmannin. Dependence on signal strength. J Biol Chem 1997;272:27665–27670.PubMedCrossRefGoogle Scholar
  89. 89.
    Bisotto S, Fixman ED. Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor. Biochem J 2001;360:77–85.PubMedCrossRefGoogle Scholar
  90. 90.
    Yu CF, Roshan B, Liu ZX, Cantley LG. ERK regulates the hepatocyte growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J Biol Chem 2001;276:32552–3558.PubMedCrossRefGoogle Scholar
  91. 91.
    Yu CF, Liu ZX, Cantley LG. ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J Biol Chem 2002;277:19382–19388.PubMedCrossRefGoogle Scholar
  92. 92.
    Moelling K, Schad K, Bosse M, Zimmermann S, Schweneker M. Regulation of Raf-Akt Cross-talk. J Biol Chem 2002;277:31099–31106.PubMedCrossRefGoogle Scholar
  93. 93.
    Park HS, Kim MS, Huh SH, et al. Akt (protein kinase B) negatively regulates SEK1 by means of protein phosphorylation. J Biol Chem 2002;277:2573–2578.PubMedCrossRefGoogle Scholar
  94. 94.
    Fujishiro M, Gotoh Y, Katagiri H, et al. Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes. Mol Endocrinol 2003;17:487–497.PubMedCrossRefGoogle Scholar
  95. 95.
    Kim JW, Lee JE, Kim MJ, Cho EG, Cho SG, Choi EJ. Glycogen synthase kinase 3 beta is a natural activator of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1). J Biol Chem 2003;278:13995–14001.PubMedCrossRefGoogle Scholar
  96. 96.
    Lee YH, Giraud J, Davis RJ, White MF. c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J Biol Chem 2003;278: 2896–2902.PubMedCrossRefGoogle Scholar
  97. 97.
    Yamamoto-Honda R, Tobe K, Kaburagi Y, et al. Upstream mechanisms of glycogen synthase activation by insulin and insulin-like growth factor-I. Glycogen synthase activation is antagonized by wortmannin or LY294002 but not by rapamycin or by inhibiting p21ras. J Biol Chem 1995;270:2729–2734.PubMedCrossRefGoogle Scholar
  98. 98.
    Nakamura K, Zhou CJ, Parente J, Chew CS. Parietal cell MAP kinases: multiple activation pathways. Am J Physiol 1996;271:G640–G649.PubMedGoogle Scholar
  99. 99.
    Scheid MP, Duronio V. Phosphatidylinositol 3-OH kinase activity is not required for activation of mitogen-activated protein kinase by cytokines. J Biol Chem 1996; 271:18134–18139.PubMedCrossRefGoogle Scholar
  100. 100.
    Cross DA, Alessi DR, Vandenheede JR, McDowell HE, Hundal HS, Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J 1994;303:21–26.PubMedGoogle Scholar
  101. 101.
    Standaert ML, Bandyopadhyay G, Farese RV. Studies with wortmannin suggest a role for phosphatidylinositol 3-kinase in the activation of glycogen synthase and mitogen-activated protein kinase by insulin in rat adipocytes: comparison of insulin and protein kinase C modulators. Biochem Biophys Res Commun 1995; 209:1082–1088.PubMedCrossRefGoogle Scholar
  102. 102.
    Marra F, Pinzani M, DeFranco R, Laffi G, Gentilini P. Involvement of phosphatidylinositol 3-kinase in the activation of extracellular signal-regulated kinase by PDGF in hepatic stellate cells. FEBS Lett 1995;376:141–145.PubMedCrossRefGoogle Scholar
  103. 103.
    Zimmermann S, Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 1999;286:1741–1744.PubMedCrossRefGoogle Scholar
  104. 104.
    Rommel C, Clarke BA, Zimmermann S, et al. Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt. Science 1999;286:1738–1741.PubMedCrossRefGoogle Scholar
  105. 105.
    Hirosumi J, Tuncman G, Chang L, et al. A central role for JNK in obesity and insulin resistance. Nature 2002;420:333–336.PubMedCrossRefGoogle Scholar
  106. 106.
    Zhang ZY, Zhou B, Xie L. Modulation of protein kinase signaling by protein phosphatases and inhibitors. Pharmacol Ther 2002;93:307–317.PubMedCrossRefGoogle Scholar
  107. 107.
    Asante-Appiah E, Kennedy BP. Protein tyrosine phosphatases: the quest for negative regulators of insulin action. Am J Physiol 2003;284:E663–E670.Google Scholar
  108. 108.
    Wu C, Sun M, Liu L, Zhou GW. The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene 2003;306:1–12.PubMedCrossRefGoogle Scholar
  109. 109.
    Elchebly M, Cheng A, Tremblay ML. Modulation of insulin signaling by protein tyrosine phosphatases. J Mol Med 2000;78:473–482.PubMedCrossRefGoogle Scholar
  110. 110.
    Tonks NK, Diltz CD, Fischer EH. Characterization of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem 1988;263:6731–6737.PubMedGoogle Scholar
  111. 111.
    Charbonneau H, Tonks NK, Kumar S, et al. Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins. Proc Natl Acad Sci USA 1989;86:5252–5256.PubMedCrossRefGoogle Scholar
  112. 112.
    Frangioni JV, Oda A, Smith M, Salzman EW, Neel BG. Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets. EMBO J 1993;12:4843–4856.PubMedGoogle Scholar
  113. 113.
    Elchebly M, Payette P, Michaliszyn E, et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 1999;283:1544–1548.PubMedCrossRefGoogle Scholar
  114. 114.
    Salmeen A, Andersen JN, Myers MP, Tonks NK, Barford D. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B. Mol Cell 2000;6:1401–1412.PubMedCrossRefGoogle Scholar
  115. 115.
    Seely BL, Staubs PA, Reichart DR, et al. Protein tyrosine phosphatase 1B interacts with the activated insulin receptor. Diabetes 1996;45:1379–1385.PubMedCrossRefGoogle Scholar
  116. 116.
    Bandyopadhyay D, Kusari A, Kenner KA, et al. Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin. J Biol Chem 1997;272:1639–1645.PubMedCrossRefGoogle Scholar
  117. 117.
    Mahadev K, Zilbering A, Zhu L, Goldstein BJ. Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade. J Biol Chem 2001;276:21938–21942.PubMedCrossRefGoogle Scholar
  118. 118.
    Wu X, Hoffstedt J, Deeb W, et al. Depot-specific variation in protein-tyrosine phosphatase activities in human omental and subcutaneous adipose tissue: a potential contribution to differential insulin sensitivity. J Clin Endocrinol Metab 2001; 86:5973–5980.PubMedCrossRefGoogle Scholar
  119. 119.
    Dadke S, Kusari A, Kusari J. Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor. Mol Cell Biochem 2001;221:147–154.PubMedCrossRefGoogle Scholar
  120. 120.
    Ravichandran LV, Chen H, Li Y, Quon MJ. Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor. Mol Endocrinol 2001;15:1768–1780.PubMedCrossRefGoogle Scholar
  121. 121.
    Tao J, Malbon CC, Wang H Y. Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo. J Biol Chem 2001;276: 29520–29525.PubMedCrossRefGoogle Scholar
  122. 122.
    Liu F, Chernoff J. Protein tyrosine phosphatase 1B interacts with and is tyrosine phosphorylated by the epidermal growth factor receptor. Biochem J 1997;327: 139–145.PubMedGoogle Scholar
  123. 123.
    Bjorge JD, Pang A, Fujita DJ. Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines. J Biol Chem 2000;275:41439–41446.PubMedCrossRefGoogle Scholar
  124. 124.
    Milarski KL, Saltiel AR. Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein kinase by insulin. J Biol Chem 1994;269:21239–21243.PubMedGoogle Scholar
  125. 125.
    Bennett AM, Hausdorff SF, O’Reilly AM, Freeman RM, Neel BG. Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression. Mol Cell Biol 1996;16:1189–1202.PubMedGoogle Scholar
  126. 126.
    Chen H, Wertheimer SJ, Lin CH, et al. Protein-tyrosine phosphatases PTP1B and syp are modulators of insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. J Biol Chem 1997;272:8026–8031.PubMedCrossRefGoogle Scholar
  127. 127.
    Ouwens DM, van der Zon GC, Maassen JA. Modulation of insulin-stimulated glycogen synthesis by Src homology phosphatase 2. Mol Cell Endocrinol 2001;175:131–140.PubMedCrossRefGoogle Scholar
  128. 128.
    Nichols A, Camps M, Gillieron C, et al. Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3. J Biol Chem 2000;275: 24613–24621.PubMedCrossRefGoogle Scholar
  129. 129.
    Masuda K, Shima H, Watanabe M, Kikuchi K. MKP-7, a novel mitogen-activated protein kinase phosphatase, functions as a shuttle protein. J Biol Chem 2001;276: 39002–39011.PubMedCrossRefGoogle Scholar
  130. 130.
    Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J 2000;14:6–16.PubMedGoogle Scholar
  131. 131.
    Sim AT, Baldwin ML, Rostas JA, Holst J, Ludowyke RI. The role of serine/threonine protein phosphatases in exocytosis. Biochem J 2003;373:641–659.PubMedCrossRefGoogle Scholar
  132. 132.
    Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 1989;58:453–508.PubMedCrossRefGoogle Scholar
  133. 133.
    Winder DG, Sweatt JD. Roles of serine/threonine phosphatases in hippocampal synaptic plasticity. Nat Rev Neurosci 2001;2:461–474.PubMedCrossRefGoogle Scholar
  134. 134.
    Klumpp S, Krieglstein J. Serine/threonine protein phosphatases in apoptosis. Curr Opin Pharmacol 2002;2:458–462.PubMedCrossRefGoogle Scholar
  135. 135.
    Brady MJ, Saltiel AR. The role of protein phosphatase-1 in insulin action. Recent Prog Horm Res 2001;56:157–173.PubMedCrossRefGoogle Scholar
  136. 136.
    Keyse SM. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol 2000;12:186–192.PubMedCrossRefGoogle Scholar
  137. 137.
    Birle D, Bottini N, Williams S, Huynh H, deBelle I, Adamson E, Mustelin T. Negative feedback regulation of the tumor suppressor PTEN by phosphoinosi-tide-induced serine phosphorylation. J Immunol 2002;169:286–291.PubMedGoogle Scholar
  138. 138.
    Ross AH, Baltimore D, Eisen HN. Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature 1981; 294:654–656.PubMedCrossRefGoogle Scholar
  139. 139.
    Fabbro D, Parkinson D, Matter A. Protein tyrosine kinase inhibitors: new treatment modalities? Curr Opin Pharmacol 2002;2:374–381.PubMedCrossRefGoogle Scholar
  140. 140.
    Toledo LM, Lydon NB, Elbaum D. The structure-based design of ATP-site directed protein kinase inhibitors. Curr Med Chem 1999;6:775–805.PubMedGoogle Scholar
  141. 141.
    Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 1995;270:27489–27494.PubMedCrossRefGoogle Scholar
  142. 142.
    Favata MF, Horiuchi KY, Manos EJ, et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 1998;273:18623–18632.PubMedCrossRefGoogle Scholar
  143. 143.
    Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000;351:95–105.PubMedCrossRefGoogle Scholar
  144. 144.
    Karihaloo A, O’Rourke DA, Nickel C, Spokes K, Cantley LG. Differential MAPK pathways utilized for HGF-and EGF-dependent renal epithelial morphogenesis. J Biol Chem 2001;276:9166–9173.PubMedCrossRefGoogle Scholar
  145. 145.
    Cuenda A, Rouse J, Doza YN, et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995;364:229–233.PubMedCrossRefGoogle Scholar
  146. 146.
    Eyers PA, Craxton M, Morrice N, Cohen P, Goedert M. Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution. Chem Biol 1998;5:321–328.PubMedCrossRefGoogle Scholar
  147. 147.
    Lali FV, Hunt AE, Turner SJ, Foxwell BM. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in inter-leukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem 2000;275:7395–7402.PubMedCrossRefGoogle Scholar
  148. 148.
    Wang L, Gout I, Proud CG. Cross-talk between the ERK and p70 S6 kinase (S6K) signaling pathways. MEK-dependent activation of S6K2 in cardiomyocytes. J Biol Chem 2001;276:32670–32677.PubMedCrossRefGoogle Scholar
  149. 149.
    Frantz B, Klatt T, Pang M, et al. The activation state of p38 mitogen-activated protein kinase determines the efficiency of ATP competition for pyridinylimidazole inhibitor binding. Biochemistry 1998;37:13846–13853.PubMedCrossRefGoogle Scholar
  150. 150.
    Bennett BL, Sasaki DT, Murray BW, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 2001;98: 13681–13686.PubMedCrossRefGoogle Scholar
  151. 151.
    Han Z, Boyle DL, Chang L, et al. c-Jun N-terminal kinase is required for metal-loproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001;108:73–81.PubMedGoogle Scholar
  152. 152.
    Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J 2003;371:199–204.PubMedCrossRefGoogle Scholar
  153. 153.
    Blake RA, Broome MA, Liu X, et al. SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol Cell Biol 2000;20:9018–9027.PubMedCrossRefGoogle Scholar
  154. 154.
    Hanke JH, Gardner JP, Dow RL, et al. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck-and FynT-dependent T cell activation. J Biol Chem 1996;271:695–701.PubMedCrossRefGoogle Scholar
  155. 155.
    Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science 1992;257:973–977.PubMedCrossRefGoogle Scholar
  156. 156.
    Kuo CJ, Chung J, Fiorentino DF, Flanagan WM, Blenis J, Crabtree GR. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 1992;358:70–73.PubMedCrossRefGoogle Scholar
  157. 157.
    Hidalgo M, Rowinsky EK. The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene 2000;19:6680–6686.PubMedCrossRefGoogle Scholar
  158. 158.
    Toullec D, Pianetti P, Coste H, et al. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 1991;266: 15771–15781.PubMedGoogle Scholar
  159. 159.
    Davis PD, Hill CH, Keech E, et al. Potent selective inhibitors of protein kinase C. FEBS Lett 1989;259:61–63.PubMedCrossRefGoogle Scholar
  160. 160.
    Gordge PC, Ryves WJ. Inhibitors of protein kinase C. Cell Signal 1994;6: 871–882.PubMedCrossRefGoogle Scholar
  161. 161.
    Martiny-Baron G, Kazanietz MG, Mischak H, et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J Biol Chem 1993;268: 9194–9197.PubMedGoogle Scholar
  162. 162.
    Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidyl-inositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269:5241–5248.PubMedGoogle Scholar
  163. 163.
    Stein RC. Prospects for phosphoinositide 3-kinase inhibition as a cancer treatment. Endocr Relat Cancer 2001;8:237–248.PubMedCrossRefGoogle Scholar
  164. 164.
    Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC Jr, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J 1996;15:5256–5267.PubMedGoogle Scholar
  165. 165.
    Izzard RA, Jackson SP, Smith GC. Competitive and noncompetitive inhibition of the DNA-dependent protein kinase. Cancer Res 1999;59:2581–2586.PubMedGoogle Scholar
  166. 166.
    Wang D, Sul HS. Insulin stimulation of the fatty acid synthase promoter is mediated by the phosphatidylinositol 3-kinase pathway. Involvement of protein kinase B/Akt. J Biol Chem 1998;273:25420–25426.PubMedCrossRefGoogle Scholar
  167. 167.
    Kitamura T, Ogawa W, Sakaue H, et al. Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol 1998;18:3708–3717.PubMedGoogle Scholar
  168. 168.
    House C, Kemp BE. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 1987;238:1726–1728.PubMedCrossRefGoogle Scholar
  169. 169.
    Yeh P, Perricaudet M. Advances in adenoviral vectors: from genetic engineering to their biology. FASEB J 1997;11:615–623.PubMedGoogle Scholar
  170. 170.
    He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998;95:2509–2514.PubMedCrossRefGoogle Scholar
  171. 171.
    Becker TC, Noel RJ, Coats WS, et al. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 1994;43:161–189.PubMedCrossRefGoogle Scholar
  172. 172.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–811.PubMedCrossRefGoogle Scholar
  173. 173.
    Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttran-scriptional gene silencing in plants. Science 1999;286:950–952.PubMedCrossRefGoogle Scholar
  174. 174.
    Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101:25–33.PubMedCrossRefGoogle Scholar
  175. 175.
    Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev 2001;15:188–200.PubMedCrossRefGoogle Scholar
  176. 176.
    McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002;3:737–747.PubMedCrossRefGoogle Scholar
  177. 177.
    Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000;403:901–906.PubMedCrossRefGoogle Scholar
  178. 178.
    Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000; 408:86–89.PubMedCrossRefGoogle Scholar
  179. 179.
    Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 2001;106:23–34.PubMedCrossRefGoogle Scholar
  180. 180.
    Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003;4:457–467.PubMedCrossRefGoogle Scholar
  181. 181.
    Baglioni C, Nilsen TW. Mechanisms of antiviral action of interferon. Interferon. 1983;5:23–42.PubMedGoogle Scholar
  182. 182.
    Williams BR. Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochem Soc Trans 1997;25:509–513.PubMedGoogle Scholar
  183. 183.
    Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001;98:9742–9747.PubMedCrossRefGoogle Scholar
  184. 184.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–498.PubMedCrossRefGoogle Scholar
  185. 185.
    McManus MT, Haines BB, Dillon CP, et al. Small interfering RNA-mediated gene silencing in T lymphocytes. J Immunol 2002;169:5754–5760.PubMedGoogle Scholar
  186. 186.
    Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550–553.PubMedCrossRefGoogle Scholar
  187. 187.
    Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002;20:500–555.PubMedGoogle Scholar
  188. 188.
    Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002;20:497–500.PubMedCrossRefGoogle Scholar
  189. 189.
    Paul CP, Good PD, Winer I, Engelke DR. Effective expression of small interfering RNA in human cells. Nat Biotechnol 2002;20:505–508.PubMedCrossRefGoogle Scholar
  190. 190.
    Sui G, Soohoo C, Affarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002;99: 5515–5520.PubMedCrossRefGoogle Scholar
  191. 191.
    Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of shortinterfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002;99:6047–6052.PubMedCrossRefGoogle Scholar
  192. 192.
    Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA 2002;99:1443–1448.PubMedCrossRefGoogle Scholar
  193. 193.
    Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for mutations and chemical modifications in a siRNA. Nucl Acids Res 2003;31:589–595.PubMedCrossRefGoogle Scholar
  194. 194.
    McManus MT, Petersen CP, Haines BB, Chen J, Sharp PA. Gene silencing using micro-RNA designed hairpins. RNA 2002;8:842–850.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Sang K. Kim
    • 1
  • Kimberley J. Woodcroft
    • 1
  • Raymond F. Novak
    • 1
  1. 1.Institute of Environmental Health SciencesWayne State UniversityDetroit

Personalised recommendations