PTPe (RPTPe and Cyt-PTPe)
Phosphorylation of tyrosine residues in proteins is one of the better-studied molecular mechanisms for regulating protein structure and function and with it – the function of cells and organisms. Tyrosine phosphorylation is a reversible process that is controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The numbers of PTKs and PTPs are similar and small relative to the numbers of their potential substrates; there are 81 and 85 genes that yield active, protein-specific PTPs and PTKs, respectively (Alonso et al. 2004). As a result, individual PTPs and PTKs each target multiple substrates and fulfill distinct roles in different physiological systems. Although PTPs were first described in molecular terms in 1988, about a decade after PTKs, both protein superfamilies are now recognized as critical regulators of protein phosphorylation and cellular physiology (Alonso et al. 2004).
The several dozen members of the PTP superfamily can be subdivided into smaller groups based on their structures and substrate specificities (Andersen et al. 2001b; Alonso et al. 2004). The first subfamily of PTPs that was identified and characterized was the “classical” tyrosine-specific subfamily of PTPs. This subfamily is now known to contain 38 genes; 21 of these encode receptor-type products that are integral membrane proteins, while the remaining 17 encode non-receptor-type proteins. Use of alternative promoters, alternative splicing, and posttranslational processing are fairly common among PTPs, hence the 38 “classical” PTP genes give rise to a larger number of protein products. All members of this family contain one or two PTP domains that contain the PTP signature motif “(I/V)HCSXGXGR(S/T)G”; in PTPs that contain two PTP domains, typically only the N-terminal of the two is catalytically active (Andersen et al. 2001b; Alonso et al. 2004). Studies conducted during the past two decades have shown that PTPs participate in regulating many distinct physiological processes, that they target specific substrates, and that they can activate or inhibit signaling processes in a context-dependent manner (Tonks 2006; Hendriks et al. 2008).
Protein tyrosine phosphatase Epsilon (PTPe), which is the focus of this review, was first identified in 1990 as a receptor-type PTP within the so-called type IV subfamily of the “classical” family of PTPs (Krueger et al. 1990). Subsequent work revealed that there is a single PTPe gene that gives rise to at least two mRNA and four protein species. More recent work, which is outlined below, has described roles for PTPe in regulating various processes at the levels of the cell and the organism.
PTPe: The Gene and its Protein Products
The cell- and tissue-specificity of the two promoters of the PTPRE gene differ significantly to the point where RPTPe and cyt-PTPe are rarely co-expressed in the same cell type. RPTPe is expressed predominantly in neurons, testes, and lungs, while cyt-PTPe is found mainly in hematopoietic cells (B- and T-lymphocytes, macrophages, erythrocytes), osteoclasts, Schwann cells, and muscle cells. Along with their distinct subcellular localization, their different expression patterns among cell types argue strongly that RPTPe and cyt-PTPe are physiologically nonequivalent.
Regulation of PTPe Activity
Expression: The divergent expression patterns of the two promoters of the PTPRE gene among cells types and tissues and their distinct localization patterns within cells ensure that RPTPe and cyt-PTPe are present at specific locations and encounter particular substrates. Importantly, the two promoters also differ in their ability to respond to physiological signals. Little is known about how the activity of the P1 promoter, which drives expression of RPTPe, is controlled. In contrast, the P2 promoter is readily activated by growth factors and activators of mitogenic signaling, such as serum, TPA, EGF, and basic FGF. Differentiation of human promyelocytic leukemia HL60 cells with TPA causes a massive increase in cyt-PTPe expression, while treatment of M1 mouse myeloid leukemia cells with IL6 increases cyt-PTPe expression and reduces expression of RPTPe (Elson and Leder 1995a; Tanuma et al. 1999).
Oxidation: The signature motif (I/V)HCSXGXGR(S/T)G present in PTPs contains at its core a cysteine residue, which is essential for dephosphorylation to occur. Oxidizing this residue by, for example, reactive oxygen species that are produced during the normal course of signaling processes is sufficient to abolish PTP catalytic activity (den Hertog et al. 2008). All forms of PTPe are susceptible to oxidation, which inhibits their activity (Toledano-Katchalski et al. 2003).
Dimerization: Dimerization of PTPs has been suggested to inhibit their catalytic activity; this sets PTPs apart from PTKs, where dimerization typically stabilizes the active form of the enzyme (den Hertog et al. 2008). RPTPe and cyt-PTPe undergo spontaneous inhibitory dimerization and higher-order aggregation in cells. In the case of cyt-PTPe, dimerization can be induced by physiological signals, such as increased oxidative stress and following activation of the EGF receptor (Toledano-Katchalski et al. 2003).
Phosphorylation: A major phosphorylation site exists at the C-terminus of PTPe (Y695 in RPTPe = Y638 in cyt-PTPe). This site undergoes phosphorylation in various physiological situations, including in the presence of Neu in mouse mammary tumor cells (Berman-Golan and Elson 2007) and following activation of integrins in osteoclasts (Granot-Attas et al. 2009). Phosphorylation most likely does not affect the specific activity of PTPe (Berman-Golan and Elson 2007). It does, however, drive the phosphatase to activate Src in osteoclasts and in mammary tumor cells, probably by allowing phosphorylated PTPe to bind the Src SH2 domain, thus releasing the inhibitory interaction between this domain and Y527 of the kinase (Berman-Golan and Elson 2007; Granot-Attas et al. 2009). C-terminal phosphorylation of PTPe also promotes its association with other molecules, as in its inhibitory association with tubulin (Sines et al. 2007).
Cleavage: RPTPe, cyt-PTPe, and p67 can undergo proteolytic processing by calpain to generate p65, an N-terminally-truncated form of PTPe that is catalytically active and entirely cytosolic (Gil-Henn et al. 2001). Processing of RPTPe or of cyt-PTPe, which are entirely or significantly membrane-associated proteins, effectively removes PTPe from the cell membrane and may control its access to substrates.
Other mechanisms: RPTPe possesses an extracellular domain, which suggests that its activity or physiological role may be affected by binding of extracellular ligands. However, as is the case with most RPTPs, no extracellular ligands of RPTPe are known, and the functional role of the extracellular domain of this PTP is not clear. RPTPe also undergoes massive glycosylation, but the roles of this modification, if any, are also unknown.
Physiological Roles of PTPe
PTKs often activate signaling processes, and many are bona fide oncogenes. As a result, PTPs are often perceived as inhibitors of signaling processes and of malignant transformation. While this is true in many instances, in several cases, including that of PTPe, PTPs can activate signaling processes and support malignant transformation. Moreover, such roles are often specific for a given physiological system; a particular PTP may support signaling and transformation in one context but inhibit it in another. Along these lines, RPTPe is expressed at high levels in mouse mammary tumors initiated in vivo by transgenic Neu or Ras proteins (Elson and Leder 1995b), suggesting that RPTPe participates in the transformation processes induced by these two oncogenes. In support of this conclusion, overexpression of RPTPe in mouse mammary glands leads to massive mammary hyperplasia and associated tumorigenesis (Elson 1999). At the molecular level, RPTPe activates the Src PTK in mammary tumors initiated by Neu, by dephosphorylating Src at its inhibitory Y527. Activation of Src is important for these cells to manifest their full malignant properties; in mammary tumors initiated by Neu in mice lacking PTPe (EKO mice), Src is less active and the transformed phenotype of the resulting mammary tumor cells is weakened (Gil-Henn and Elson 2003; Berman-Golan and Elson 2007). On the other hand, overexpression of cyt-PTPe (but not of RPTPe) in M1 murine leukemia cells inhibited JAK-STAT signaling that was induced by interleukins 6 or 10, and led to reduced tumorigenicity of these cells following their implantation in SCID or in nude mice (Tanuma et al. 2001; Tanuma et al. 2003). The various protein forms of PTPe can therefore either support or inhibit signaling events in a context- and system-specific manner.
Myelination in the Nervous System
Experimental evidence has shown that PTPe helps regulate myelination in the nervous system. Cyt-PTPe is expressed in Schwann cells, which are responsible for axon myelination in the peripheral nervous system. In mice that genetically lack PTPe, myelination of axons in the sciatic nerve is significantly delayed, indicating that this form of PTPe supports the myelination process (Peretz et al. 2000). Molecular studies have established that the delayed-rectifier, voltage-gated potassium channels Kv2.1 and Kv1.5 are substrates of cyt-PTPe in Schwann cells, and that dephosphorylation by cyt-PTPe downregulates channel activity. In agreement, Kv2.1 and Kv1.5 are hyper-phosphorylated, and the activity of Kv channels in general is elevated in EKO Schwann cells, suggesting that loss of cyt-PTPe in Schwann cells affects their function by dysregulating Kv channel activity (Peretz et al. 2000). In separate studies, expression of PTPe, presumably RPTPe, was significantly up-regulated during differentiation of CG4 progenitor cells into oligodendrocytes, the cells that drive myelination in the central nervous system. Expression in mice of an inactive mutant of RPTPe under the direction of the myelin protein 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter delayed myelination of the optic nerve, most likely due to dominant-negative effects of the transgene (Muja et al. 2004). The limited scope of the phenotype in this case suggests that lack of RPTPe does not significantly affect the myelination capability of most oligodendrocytes.
Macrophages, Osteoclasts, and Bone Degradation
Cyt-PTPe is expressed in cells of various hematopoietic lineages, including in cells of the monocyte lineage that gives rise to macrophages and osteoclasts. Macrophages from mice lacking PTPe are defective in their ability to mount a respiratory burst in response to exposure to lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF alpha). Bone marrow cells from these mice also produce more interleukin-10 and less TNF alpha in response to LPS treatment (Sully et al. 2001). These findings indicate that cyt-PTPe plays an important role in regulating macrophage activity, although the molecular basis for this remains unknown at present.
Cyt-PTPe also plays a major role in regulating the function of osteoclasts, the resident cells that degrade bone. Bone mass is regulated by the opposing activities of osteoblasts, which produce bone matrix, and osteoclasts, which degrade it. Both cell types coexist and function in close proximity, ensuring that the resulting bone is of the proper mass and physical properties. Mice lacking PTPe exhibit increased amounts of bone that are secondary to reduced activity of their osteoclasts. Osteoclasts lacking cyt-PTPe do not adhere to bone well in vivo and display significant defects in the structure, organization, and stability of podosomes, the adhesion structures of these cells (Granot-Attas et al. 2009). At the molecular level, cyt-PTPe helps dephosphorylate and activate Src downstream of integrins, which are activated when osteoclasts make physical contact with bone or matrix. Lack of cyt-PTPe results in reduced Src activation in osteoclasts; normal Src activity can be restored by expression of cyt-PTPe in the cells. Importantly, the defects in podosomal organization and stability can be rescued in PTPe-deficient osteoclasts not only by expressing cyt-PTPe but also by expressing Src, which functions in this system downstream of cyt-PTPe. This finding demonstrates that the integrin-cyt-PTPe-Src axis is critical for proper structure and function of osteoclasts (Granot-Attas et al. 2009).
Insulin Receptor Signaling, Glucose Homeostasis and Body Weight Regulation
A number of studies have suggested that PTPe negatively regulates the activity of the insulin receptor PTK. Accordingly, RPTPe, but not cyt-PTPe, has been shown to down-regulate the insulin receptor in baby hamster kidney (BHK) cells (Andersen et al. 2001a). Later studies showed that expression of exogenous RPTPe down-regulates the insulin receptor in primary hepatocytes, possibly by targeting the insulin receptor itself. Expression of the phosphatase also reduced the extent of activation by insulin of downstream signaling molecules, such as ERK, AKT, and GSK3 (Nakagawa et al. 2005). A third study showed that cyt-PTPe negatively regulates the insulin receptor in muscle cells. Treating L6 skeletal muscle cells with insulin induced association of the insulin receptor with cyt-PTPe, while expression of exogenous cyt-PTPe resulted in decreased phosphorylation of the insulin receptor, IRS-1, AKT and GSK3, and decreased glucose uptake. In agreement, opposite results were obtained when expression of endogenous cyt-PTPe was inhibited in these cells. Phosphorylation of the insulin receptor and or IRS-1 were also increased in primary muscle cells from PTPe-deficient mice (Aga-Mizrachi et al. 2008). Examination of mice genetically lacking PTPe revealed improved glucose clearance in both lean and obese mice, particularly in males, indicating that PTPe plays a role in regulating glucose homeostasis on the level of the intact organism (Rousso-Noori et al. 2011). RPTPe and cyt-PTPe are therefore inhibitors of insulin receptor signaling, although more studies are required to determine whether organ-specific roles exist for various isoforms of PTPe.
Female mice lacking PTPe are leptin hypersensitive and are resistant to weight gain that normally follows physiological challenges, such as a high-fat diet. This is most likely caused by down-regulation of leptin receptor signaling by RPTPe in the hypothalamus; RPTPe performs this role by directly dephosphorylating and inhibiting Jak2, a tyrosine kinase that is activated downstream of the leptin receptor and which plays a key role in leptin signaling (Rousso-Noori et al. 2011).
PTPe is expressed in endothelial cells (Thompson et al. 2001; Nakagawa et al. 2004). The role of PTPe in these cells appears to be isoform-specific, since expression of RPTPe activated Src and stimulated migration and survival of porcine aortic endothelial cells, while expression of cyt-PTPe had the opposite effect (Thompson et al. 2001; Nakagawa et al. 2004). RPTPe is expressed in erythrocytes; erythrocytes of EKO mice exhibit abnormal morphology, increased calcium-activated K+ channel activity, and increased activity of the Src family PTKs Fyn and Yes. These findings indicate that RPTPe plays an important role in erythrocyte physiology and provide an example where this PTP down-regulates, instead of activating, PTKs of the Src family (De Franceschi et al. 2008). Finally, RPTPe is expressed in bone marrow–derived mast cells (BMMC), where it participates in downregulating FceRI-mediated mast cell function (Akimoto et al. 2009). RPTPe most likely targets the Syk PTK downstream of the FceRI receptor, leading ultimately to decreased calcium mobilization and MAPK activation. Accordingly, EKO mice display elevated levels of passive systemic anaphylaxis induced by antigen and IgE. EKO mice contain normal numbers of mast cells, indicating that lack of PTPe affects the function, but not production, of these cells (Akimoto et al. 2009).
Like other PTPs and PTKs, PTPe is a multifaceted participant of signaling processes. The single PTPe gene produces several distinct protein isoforms, which act in different cell types, in distinct areas within a given cell, and target various substrates. It is therefore not surprising that PTPe supports the transduced signal in some cases while inhibiting it in others. It is very well established that dysregulation of protein phosphorylation is a cause for disease in human beings; indeed, increasing numbers of novel drugs are becoming available, which treat a particular disease by specifically targeting molecules that play key roles in its etiology. Design of reagents that target a particular PTP for medical gain remains a worthy, albeit elusive, goal. For this to occur, continued studies that will characterize in full the functions of PTPe and of other PTPs at the molecular, cellular, and whole-organism levels are required. Studies of this type should also address functional redundancies between PTPs, making it clear which other PTPs should also be targeted to obtain a particular outcome, and which PTPs perform opposite roles in this context and should not be targeted. A related challenge is to fully decipher the repertoire of proteins that PTPe acts upon in cells. Traditional biochemical methods address this issue at the level of the individual substrate protein, while advanced proteomic techniques, such as mass spectrometry, may provide unbiased data on a cell-wide basis.