Protein Kinase D Signaling*

A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as one of the fundamental mechanisms of signal transduction in animal cells. A plethora of external signals, including hormones, neurotransmitters, growth factors, cytokines, bioactive lipids, and tastants promote the stimulation of the isoforms of the PLC family, including , , , and . PLCs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce two second messengers: inositol 1,4,5-P3, which triggers the release of Ca from internal stores, and DAG, which elicits cellular responses through a variety of effectors (1). The most prominent intracellular targets of DAG are the classic ( , , ) and novel ( , , , ) isoforms of PKC, which are differentially expressed in cells and tissues (2, 3). The mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely understood. Protein kinase D (PKD), the founding member of a new family of serine/threonine protein kinases and the subject of this minireview, occupies a unique position in the signal transduction pathways initiated by DAG and PKC. PKD not only is a direct DAG target but also lies downstream of PKCs in a novel signal transduction pathway implicated in the regulation of multiple fundamental biological processes.


PKD Family Belongs to CAMK Group
Complementary DNA clones encoding human PKD (initially called atypical PKC) and PKD from mouse were identified by two different laboratories in 1994 (4,5). Subsequently, two additional mammalian protein kinases have been identified that share extensive overall homology with PKD ( Fig. 1), termed PKD2 (6) and PKC/PKD3 (7,8).
The N-terminal regulatory portion of PKD (Fig. 1) contains a tandem repeat of zinc finger-like cysteine-rich motifs (termed the cysteine-rich domain or CRD) highly homologous to domains found in DAG/phorbol ester-sensitive PKCs and in other signaling proteins regulated by DAG, including chimerins, Ras-GRP, Munc13, and DAG kinases (see Ref. 1 for review). Accordingly, PKD binds phorbol esters with high affinity via its CRD (5,9,10). The individual cysteine-rich motifs of the CRD, referred to as cys1 and cys2 (Fig. 1), are functionally dissimilar with the cys2 motif responsible for the majority of high affinity [ 3 H]phorbol 12,13-dibutyrate binding both in vivo and in vitro (11,12). As described below, the CRD plays a critical role in mediating PKD translocation to the plasma membrane and nucleus in cells challenged with a variety of stimuli and also represses the catalytic activity of the enzyme (13).
Interposed between the CRD and the catalytic domain, PKD also contains a PH domain (14). Found in many signal transduction proteins, PH domains bind to membrane lipids as well as to other proteins (reviewed in Ref. 15). PH domains have also been determined to play an autoregulatory role in some protein kinases, including PKD. Thus, PKD mutants with deletions or with single amino acid substitutions within the PH domain are fully active (14,16), indicating that the PH domain, like the CRD, helps to maintain PKD in an inactive catalytic state.
The initial description of PKD as an atypical isoform of PKC (4) and the inclusion of PKD/PKC in reviews concerning the PKC family, which belongs to the AGC group (named for PKA, PKG, and PKC) (2, 3), contributed to a perception that PKD belongs to the PKC family. However, it was noted from the outset that the catalytic domain of PKD has highest sequence homology with myosin light chain kinase and CAMKs (5). Indeed, the three isoforms of PKD are now classified as a new protein kinase family within the CAMK group, separate from the AGC group (17). This scheme reflects the notion that the evolutionary relationship between protein kinases is most appropriately linked to their respective catalytic domain structures.
Full-length PKD isolated from multiple cell types or tissues exhibits very low catalytic activity (9) that can be stimulated by phosphatidylserine micelles and either DAG or phorbol esters (9,18,19). These early studies demonstrated that PKD is a phospholipid/DAG-stimulated serine/threonine protein kinase and implied that PKD represents a novel component of the signal transduction initiated by DAG production in their target cells (20).

PKD Activation in Intact Cells: a PKC/PKD Phosphorylation Cascade
Subsequent studies, which aimed to define the regulatory properties of PKD within intact cells, produced multiple lines of evidence that elucidated a mechanism of PKD activation distinct from the direct stimulation of enzyme activity by DAG/ phorbol ester plus phospholipids obtained in vitro. Treatment of intact cells with phorbol esters, cell-permeant DAGs, or bryostatin induced a dramatic conversion of PKD from an inactive to an active form, as shown by in vitro kinase assays performed in the absence of lipid co-activators (19,21). In all these cases, PKD activation was selectively and potently blocked by cell treatment with PKC inhibitors (e.g. GFI and Ro 31-8220) that did not directly inhibit PKD catalytic activity (19,21), suggesting that PKD activation in intact cells is mediated, directly or indirectly, through PKCs. In line with this conclusion, cotransfection of PKD with active mutant forms of "novel" PKCs (PKCs ␦, ⑀, , ) resulted in robust PKD activa-* This minireview will be reprinted in the 2005 Minireview Compendium, which will be available in January 2006. Studies from our laboratory presented in this review were supported in part by National Institutes of Health Grants DK 55003, DK 56930, and P50 CA90388. tion in the absence of cell stimulation (16,(21)(22)(23).

PKC⑀ Directly Phosphorylates PKD at Activation Loop
A critical aspect in the regulation of protein kinases that function in signaling cascades is the phosphorylation of activating residues, within the kinase catalytic domain, located in a region spanning the highly conserved sequences DFG (in kinase subdomain VII) and APE (in kinase subdomain VIII) termed the "activation loop" or "activation segment." Using two-dimensional tryptic phosphopeptide mapping of metabolically 32 P-labeled wild type and mutant forms of PKD, two key serine residues in the PKD activation loop, Ser-744 and Ser-748 in mouse PKD (Fig. 1), were identified (57,58). Whereas a PKD mutant with Ser-744 and Ser-748 altered to alanine was resistant to activation in response to cell stimulation, mutation of both sites to glutamic acid residues (to mimic phosphorylation) generated a constitutively active PKD. Single point mutants in which glutamic acid replaced Ser-744 or Ser-748 produced partly activated kinases. The properties of these mutant forms of PKD were consistent with a role of Ser-744 and Ser-748 in phosphorylation-dependent activation.
Using an antibody that recognizes PKD phosphorylated at Ser-748 and a second antibody that detects predominantly PKD phosphorylated at Ser-744, PKD activation loop phosphorylation was demonstrated in response to regulatory peptides, expression of heterotrimeric G proteins, and oxidative stress in many cell types (26, 50, 55, 59 -61). In line with the existence of a kinase cascade, Ser-744 and Ser-748 also become phosphorylated in kinase-deficient forms of PKD, indicating that PKD activation depends on transphosphorylation by an upstream kinase (e.g. PKC) rather than on PKD autophosphorylation (61). Although phosphorylation of other serine (40,57) and tyrosine (55,56) residues is likely to play a role in PKD regulation, it is clear that PKD phosphorylation at Ser-744 and Ser-748 is triggered by a vast array of stimuli in multiple cell types.
Recent studies in vitro and in vivo examined further the role of PKC⑀ as an upstream kinase in the activation loop phosphorylation of PKD. When incubated in the presence of phosphatidylserine, phorbol ester, and ATP, intact PKD slowly autophosphorylated at Ser-748. In striking contrast, addition of purified PKC⑀ to the incubation mixture induced rapid Ser-744 and Ser-748 phosphorylation, concomitant with a persistent increase in PKD catalytic activity (62). Furthermore, selective suppression of PKC⑀ expression in intact cells markedly attenuated activation loop phosphorylation induced by GPCR stimulation (30). Collectively, these studies substantiate the notion that novel PKCs directly activate PKD by activation loop phosphorylation at Ser-744 and Ser-748.

Intracellular Distribution of PKDs
PKD is present in the cytosol of unstimulated cells (27,38,41,64) and to a lesser extent in several intracellular compartments including Golgi and mitochondria (65, 66) but rapidly translocates from the cytosol to different subcellular compartments in response to receptor activation (27,38,41,64,67). Each translocation step is associated with a particular PKD domain and involves rapid and reversible interactions (Fig. 2). The first step of PKD translocation is mediated by the cys2 motif of the CRD, which binds to DAG produced at the inner leaflet of the plasma membrane as a result of PLC stimulation (27). It has also been reported that cys2 and flanking sequences can directly bind to G␣ q (68). In contrast, the cys1 recruits PKD to the Golgi apparatus (69). The second step, i.e. reverse translocation from the plasma membrane to the cytosol, requires the PKC-dependent phosphorylation of PKD activation loop Ser-744 and Ser-748 (27). Phosphorylated PKD is then imported, via its cys2 motif, into the nucleus, where it transiently accumulates before being exported to the cytosol through a CRM1-dependent nuclear export pathway that requires the PH domain of PKD (64).
Antigen receptor engagement of B cells and mast cells induces rapid translocation of PKD from the cytosol to the plasma membrane (38,41). The plasma membrane translocation promoted by antigen receptor engagement is reversible and does not appear to involve the nuclear compartment (38). These different observations emphasize the notion that in addition to the structural determinants present in PKD, other factors including cell context, stimulus, and scaffolding proteins also influence its intracellular distribution. In this context, the protein kinase A anchoring protein (AKAP-Lbc), which possesses Rho-specific guanine nucleotide exchange activity and is linked to G␣ 12/13 signaling, forms a multiprotein complex that includes PKD, PKC, and PKA that facilitates PKD translocation and activation (70). Previous results also demonstrated that G␣ 13 and activated Rho promote PKD activation (50,52). These findings support the notion that GPCRs utilize both G q and G 12/13 pathways to induce PKD transloca- tion and activation in their target cells.
PKD2 also undergoes reversible translocation from the cytosol to the plasma membrane in response to GPCR stimulation (71). The reverse translocation of PKD2 requires PKC activity and, as in the case of PKD, can be prevented by inhibiting the translocation of PKC⑀ (30). In contrast to PKD, active PKD2 remains predominantly in the cytoplasm after its plasma membrane dissociation.
Unlike either PKD or PKD2, PKD3 is present in the nucleus as well as the cytoplasm of unstimulated cells (8). Stimuli including GPCR agonists (e.g. neurotensin) and B cell antigen receptor engagement induce a rapid and reversible plasma membrane translocation of PKD3 (8,72). Similar to PKD, but not PKD2, GPCR activation enhances the rate of PKD3 entry into the nucleus (8). The differences in the intracellular distribution of the different PKD isoenzymes may confer the ability to execute multiple functions at distinct subcellular locations.

A Multistep Model of PKD Localization, Phosphorylation, and Catalytic Activation
The studies discussed here suggest a sequential model of PKD activation in response to rapid generation of DAG in the plasma membrane that integrates the spatial and temporal changes in PKD localization with PKD catalytic activity and multisite phosphorylation (8,30,55,61,62,64). The salient features of this model, illustrated in Fig. 2, are as follows. 1) In non-stimulated cells, PKD is in a state of very low kinase catalytic activity maintained through repression mediated by the CRD and PH domains. In these cells, the steady-state distribution of PKD results from nucleocytoplasmic shuttling in which the rate of nuclear export exceeds that of nuclear import. 2) Production of DAG induces CRD-mediated PKD translocation from the cytosol to the plasma membrane where novel PKCs are also recruited in response to DAG generation. 3) Novel PKCs, allosterically activated by DAG, transphosphorylate PKD at Ser-744 and Ser-748, thereby stabilizing the activation loop of this enzyme in an active conformation. 4) The phosphorylated and activated PKD dissociates from the plasma membrane, translocates to the cytosol, and subsequently enters into the nucleus. Thus, PKCs coordinate PKD activation by phosphorylation of Ser-744 and Ser-748 with rapid dissociation of PKD from the plasma membrane to propagate DAG-PKC signals initiated at the cell surface into the interior of the cell, including the nucleus.
Recent results suggest that a similar model could explain the regulation of the catalytic activity and intracellular distribution of PKD2 and PKD3 in response to agonist-induced DAG generation. In the framework of this model, the steady-state distribution of inactive PKD, PKD2, and PKD3 in the cytosol and nucleus results from their respective rates of nuclear import and nuclear export (8,64,71). We envisage that, as discussed above and presented in Fig. 2 for PKD, production of DAG in the plasma membrane also triggers changes in localization, phosphorylation, and catalytic activation of PKD2 and PKD3. In this way, a similar mechanism of PKD family activation can potentially generate diverse physiological responses based on the differential distribution of each isoform.

PKD Functions
The multistep model of activation shown in Fig. 2 suggests that the PKDs are well positioned to regulate membrane, cytoplasmic, and nuclear events. Indeed, it is emerging that the PKDs are implicated in the regulation of a remarkable array of fundamental biological processes, including signal transduction, membrane trafficking, and cell survival, migration, differentiation, and proliferation. PKD alters the relative activities of the JNK and ERK pathways, attenuating JNK activation and c-Jun phosphorylation in response to epidermal growth factor receptor activation (73,74) while stimulating Ras and ERK pathways (48,59,75,76). PKD regulates the budding of secretory vesicles from the trans-Golgi network (65,77), a process required for fibroblast locomotion and localized Rac1dependent leading edge activity (78). In line with an important role in cell trafficking and motility, PKD also promotes integrin recruitment to newly formed focal adhesions (79) and invasiveness of cancer cells (46,80). Furthermore, PKD plays a role in regulating apoptosis (81), T cell differentiation in transgenic models (82), re-initiation of DNA synthesis induced by phorbol esters and regulatory peptides that act via G q -coupled receptors (26,48), and cardiac hypertrophy (83).
PKD is also implicated as a mediator in stress and disease states, including human hypertrophic cardiomyopathy, the most common cause of sudden cardiac death in the young (84), Bcr-Abl-induced nuclear factor B activation in human myeloid leukemia (45), and in oxidative stress responses (54 -56). The involvement of PKDs in mediating such a diverse array of normal and abnormal biological activities in different subcellular compartments is likely to depend on the dynamic changes in their spatial and temporal localization combined with their distinct substrate specificity.
It is plausible that some of these functions are interdependent and necessary to elicit a specific cellular response. For example, the stimulatory effect of PKD on GPCR-induced cell proliferation (26) has been linked to its ability to increase the duration of the MEK/ERK/RSK pathway, leading to accumulation of immediate gene products including c-Fos that stimulate cell cycle progression (48). Recent reports have revealed that PKD directly phosphorylates class II histone deacetylase 5 (HDAC5), an enzyme that induces chromatin modifications and suppresses cardiac hypertrophy (83). PKD-mediated phosphoryl- In stimulated cells, inactive PKD translocates from the cytosol to the plasma membrane in response to DAG produced in that cellular compartment via PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. DAG also recruits to the plasma membrane and simultaneously activates novel PKCs, which then mediate the transphosphorylation and activation of PKD. Active PKD then dissociates from the plasma membrane and migrates to the cytosol and subsequently into the nuclei, where it transiently accumulates before returning to its steadystate distribution prior to cell stimulation. The broken line arrows denote steps that are not proven. Arrow direction and thickness represent the differential rates and directionality of PKD transport. PM, plasma membrane, Cyt, cytosol; Nuc, nucleus; circled P, phosphate. ation of HDAC5 neutralizes its ability to suppress cardiac hypertrophy by triggering CRM1-dependent nuclear export (83).
Although the immediate downstream targets of PKD necessary for transmitting its signals have not been fully identified, putative substrates are beginning to emerge, including the neuronal protein Kidins 220 (34,63), the Ras effector RIN1 (75), cardiac HDAC5 (83), the vanilloid receptor type 1 (35), and troponin I (33). It is increasingly apparent that the members of the PKD subfamily are key players in the regulation of cell signaling, organization, migration, apoptosis, and proliferation.

Concluding Remarks
A great deal of progress has been made in understanding the regulatory mechanisms of PKD activation and subcellular regulation and the role of novel PKCs in PKD activation loop phosphorylation. As in other cascades, inducible activation loop phosphorylation provides a mechanism of signal integration and amplification. Recent advances demonstrate an important role of the PKDs in a variety of fundamental biological processes, and the hunt for physiological substrates is well under way. In conclusion, studies of PKD thus far indicate a remarkable diversity of both its signal generation and distribution and its potential for complex regulatory interactions with multiple downstream pathways.