The serine/threonine protein kinase D (PKD) was first described in 1994. Due to some homology in its kinase domain, its ability to bind the lipid diacylglycerol (DAG), and its responsiveness to phorbol ester, PKD was originally classified as a member of the protein kinase C (PKC) family (Johannes et al. 1994). Later, it became evident that PKD represents an own group of kinases more closely related to the calcium/calmodulin-regulated (Cam) kinases (Manning et al. 2002). Initially, the PKD field focused on determining the functions of the structural elements and domains of PKD1 as well as the activation mechanisms for this kinase. With the identification of the PKD phosphorylation motif (Hutti et al. 2004) and the subsequent generation of a substrate-specific antibody (Döppler et al. 2005), optimal conditions were provided for the discovery of PKD substrates. This led to an increasing wealth of information on PKD downstream signaling, its role in various cellular processes, as well as its potential roles in health and disease. For example, it was shown that PKD has multiple functions within cells ranging from regulating Golgi organization and vesicle transport, cell proliferation, cell survival, to mitochondrial signaling to the nucleus (reviewed in Wang 2006). To date, three highly conserved isoenzymes, PKD1/PKCμ, PKD2, and PKD3/PKCν, have been described. Dependent on the cellular context, the three PKD family members may have redundant or unique functions. This is currently under intense investigation using shRNA strategies in cellular settings or in knockout and knockin animal models.
Structure of PKD Enzymes
Regulation of PKD Activity
The canonical activation pathway for PKD is binding to DAG or phorbol ester, which leads to the release of the inhibition through the regulatory domain and subsequent phosphorylation of PKD by nPKC enzymes including PKCε, PKCδ, PKCη, and PKCθ. For example, in response to growth factor signaling, after translocation to the plasma membrane via binding to DAG, PKD is activated through nPKC. PKC enzymes phosphorylate PKD in the activation loop of the kinase domain at serine residues Ser738 and Ser742 (for human PKD1). These phosphorylation steps are required for PKD to gain full activity (Rozengurt et al. 2005). Active PKD can be released from the plasma membrane after this phosphorylation or can stay attached to intracellular membranes through binding to adapter proteins (i.e., Golgi localization through binding of G-protein βγ subunits to the PH domain).
Activation of PKD1 and PKD2 in response to oxidative stress requires additional phosphorylation events. For example, at the mitochondria, the activation of PKD1 by reactive oxygen species (ROS) also is initiated through binding to DAG. However, PKD1 activation then requires further phosphorylations at several tyrosine residues through the kinases Src and Abl. Abl phosphorylates PKD1 in its PH domain at tyrosine residue Y463 (for human PKD1) and this leads to a release of the inhibitory regulatory domain. Subsequently, Src phosphorylates PKD1 at tyrosine residue Y95, generating a binding motif for PKCδ, which then phosphorylates the two activation loop serines in the kinase domain (Döppler and Storz 2007).
Activation of PKD was also linked to autophosphorylation at serine residue S910 (in human PKD1) (Matthews et al. 1999). However, there is increasing evidence that this site gets phosphorylated independently or prior to PKD1 activation loop phosphorylation, suggesting that in vivo also other kinases may be involved in the phosphorylation of this residue. S910 is localized within a PDZ (postsynaptic density-95/discs large/zonula occludens-1-binding motif) motif and its phosphorylation leads to a release of a PDZ-binding protein. This process was functionally linked to the release of vesicles from the TGN (Sanchez-Ruiloba et al. 2006).
Additional in vivo phosphorylation sites in PKD include residues S203, S208, S219, and S223 (for human PKD1), whose phosphorylation regulates binding of PKD1 to 14-3-3, a mechanism that negatively regulates the activity of the kinase (Hausser et al. 1999). The phosphorylation of these sites is most likely mediated through auto- or transphosphorylation.
Cellular Localization and Functions
Shuttling of PKD Within Cells
Canonical activation of PKD occurs at the plasma membrane. Upon generation of DAG, PKD shuttles from the cytosol to the plasma membrane where it obtains activating phosphorylations. Dependent on the stimulus, active PKD is then released into the cytosol or shuttled to the nucleus. Cytosolic PKD, for example, mediates activation of the MAP kinase cascade. Nuclear targets for PKD are histone deacetylases (HDACs), whose phosphorylation by PKD leads to nuclear export (reviewed in Wang 2006).
PKD at the Mitochondria
PKD can be localized to the mitochondria in response to either exogenously or endogenously induced oxidative stress. The mechanism of how PKD1 localizes to the mitochondria is not fully understood but requires tyrosine phosphorylations and binding to DAG. Substrates for PKD1 at the mitochondria have not been described so far. PKD requires mitochondrial activation to gain the ability to activate antiapoptotic and antioxidant signaling through the transcription factor NF-κB (Cowell et al. 2009; Storz and Toker 2003). The functions of mitochondrially activated PKD are stress resistance and cell survival.
PKD at the Golgi Compartment
A subcellular pool of PKD is localized to the trans-Golgi network (TGN) and regulates the fission of transport carriers specifically destined to the cell surface (reviewed in Ghanekar and Lowe 2005). For example, PKD1 and PKD2 regulate neurotensin secretion in pancreatic cancer cells. A kinase-dead version of PKD1 blocks the detachment of tubes with cargo for the plasma membrane from the TGN. Binding of PKD to the TGN requires DAG and recruitment to the TGN occurs via the C1a domain (Baron and Malhotra 2002). Further, PKD activation is mediated by PKCη and G-protein βγ subunits. Only a few PKD substrates at the Golgi were described so far. Phosphatidylinositol 4-phosphate (PI4-P) generated from phosphatidylinositol by PI4 kinases (PI4K) is an important lipid mediator in vesicular trafficking. Phosphorylation of the PI4 kinase PI4KIIIβ by PKD at the Golgi complex facilitates its stabilization and increases its kinase activity through binding of 14-3-3 proteins (Hausser et al. 2005). Another PKD substrate is CERT (ceramide transport), which is also involved in PKD activation at the Golgi (Fugmann et al. 2007).
PKD at the Actin Cytoskeleton
Recently, it was shown that a subcellular pool of PKD interacts with actin and once active inhibits actin reorganization at the lamellipodium (Eiseler et al. 2009). PKD isoenzymes can be activated by small Rho GTPases including RhoA, Rac, and Cdc42 (Rozengurt et al. 2005). All these Rho GTPases have major roles in regulating responses that lead to cytoskeletal reorganization involved in the regulation of actin stress fibers, focal adhesions, or cell motility, and it was shown that PKD1 regulates some of the RhoA-mediated cytoskeletal changes through phosphorylation of SSH1L and regulation of cofilin activity (Eiseler et al. 2009).
PKD in Human Disease
The in vivo functions of PKD enzymes are not well understood. So far, best studied are the roles in cardiac failure, immune cell function, and cancer, although the exact contributions of the three PKD isoforms are still ill-defined.
Role of PKD in Coronary Heart Disease
Coronary heart disease is associated with cardiac hypertrophy, which precedes cardiac failure. Cardiac hypertrophy can be mediated by altered expression of genes regulating myocyte contraction, cell metabolism, or calcium homeostasis. The transcription of such genes is regulated by histone acetylation processes. PKD1 was shown to negatively regulate the function of histone deacetylase (HDAC) enzymes resulting in increased transcriptional activity of genes that have been linked to mediating hypertrophy in mycocytes (Vega et al. 2004).
Role of PKD in Immune Cells
PKD is activated after immune cell receptor stimulation. This includes the activation of mast cell receptors and B-cell and T-cell receptors and suggests potential roles for PKD enzymes in mediating diverse immune responses. However, the roles of PKD isoforms in regulating immune functions in vivo are not well defined. Recently, the analysis of knockin and knockout animal models demonstrated that PKD2 has a major role in regulating antigen receptor-induced cytokine production and T-cell dependent antigen responses in vivo.
Role of PKD in Cancer
The functions of PKD enzymes in cancer range from tumor-initiating events, including increased DNA synthesis, proliferation, and survival signaling, to events regulating tumor cell progression, such as angiogenesis (Liou et al. 2015; Wang 2006). Moreover, PKD is activated by tumor-promoting phorbol esters and activating phosphorylations are mediated by PKCs, Src, and Abl, all kinases that previously have been implicated in various functions in cancer. Therefore, on first view PKD represents a bona fide oncogene. However, the role of PKD in processes regulating tumor cell migration and invasion are not well understood. Accumulating reports suggest that PKD1/2 activation suppresses and PKD3 increases directed cell migration and tumor cell invasion (Sanchez-Ruiloba et al. 2006; Eiseler et al. 2009). Moreover, converse results were obtained regarding the function of PKD1 in regulating cell migration in epithelial or endothelial cells, and it is possible that the effect of PKD1 on cell motility is cell type and cancer type dependent. Additionally, epigenetic silencing of PKD1 occurs in invasive breast or gastric cancers at the transcriptional levels through methylation of a CpG island in its promoter (Borges et al. 2013; Kim et al. 2008). This suggests that PKD1 expression may be needed for early tumorigenic events but is downregulated during metastatic progression.
Although intensively investigated since over 15 years, the in vivo roles of PKD enzymes are still unclear. Most of the knowledge on PKD signaling bases on cell culture experiments performed on PKD1. Because of their potential role in human cancer, PKD signaling molecules gained increasing interest as chemotherapeutic targets. However, it remains obscure if these strategies will be effective for invasive cancers since PKD enzymes in some cancers may inhibit the migration and invasion of cancer cells at multiple levels, in other cells may have a promoting function for these processes (Durand et al. 2015). In animals, adult tissues often show coexpression of the different PKD isoforms and functional redundancy between the family members has been described but is not always the case. PKD1 and PKD2 are the most closely related mammalian PKD isoforms with an approximately 86% overall identity at the amino-acid level. Within recent years, it became apparent that both share common regulatory mechanisms and have redundant functions to some stimuli and unique or even converse functions in response to others. In cancer, this becomes most apparent. For example, PKD1 contributes to early events leading to epithelial tumor cell proliferation and survival but is also a negative regulator of cell invasion. On the other hand, PKD3 seems to facilitate metastatic progression. In other diseases such as cardiac hypertrophy, where all the three PKD isoforms seem to have overlapping functions, the use of PKD-specific inhibitors may be more effective.
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