Anchoring proteins for protein kinase C: a means for isozyme selectivity

^a Department of Molecular Pharmacology, Stanford University, School of Medicine, Stanford, California 94305–5332, USA
^b Departments of Neurology and Cellular and Molecular Pharmacology, Ernest Gallo Clinic and Research Center, Program in Neuroscience, and Center for the Neurobiology of Addiction, University of California, San Francisco, California 94110, USA

	ABSTRACT

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 
Protein kinase C (PKC) isozymes comprise a family of related enzymes. There are only limited differences between these isozymes in substrate specificity or sensitivity to activators. However, there are multiple isozymes within a cell mediating isozyme-specific functions. Differential subcellular localization has been proposed to explain this specificity. When members of the PKC family are activated by lipid-derived second messengers, they translocate from one cell compartment to another. Isozyme specificity appears to be mediated in part by association of each PKC isozyme with specific anchoring proteins. This review will cover the proteins involved in the anchoring of PKC isozymes at specific subcellular sites, the domains in the PKC isozymes that mediate protein–protein interaction with isozyme-specific anchoring proteins, and identification of peptides that interfere with or promote these protein–protein interactions, thus altering the localization and function of individual isozymes.—Mochly-Rosen, D., Gordon, A. S. Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J. 12, 35–42 (1998)

Key Words: PKC • RACK • RICK • localization

	INTRODUCTION

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 
PROTEIN KINASE C (PKC)² is a family of protein kinases that undergoes translocation from one intracellular compartment to another when activated by neurotransmitters, hormones, and growth factors. Most members of this family depend for their activation on phosphatidylserine (PS), diacylglycerol (DG), and, to different extents, on calcium and other lipid second messengers (1, 2). The PKC family of isozymes can be divided into at least three subfamilies based on their homology and sensitivity to activators ( Fig. 1). Members of the classical or cPKC subfamily—, ßI, ßII, and PKC—contain four homologous domains (C1, C2, C3, and C4) interspaced with isozyme-unique (variable or V) domains, and require calcium, PS, and DG or phorbol esters for activation. Members of the novel or nPKC subfamily—, , , and PKC—lack the C2 homologous domain and do not require calcium for activation. Finally, members of the atypical or aPKC subfamily, and /PKC, lack both the C2 and one-half of the C1 homologous domains and are insensitive to diacylglycerol, phorbol esters, and calcium. A recently described PKC isozyme, µPKC or PKD, does not fit into any of the major PKC subfamilies. It is phospholipid-dependent, calcium-insensitive, and activated by phorbol esters. However, it contains two unique hydrophobic domains in the amino-terminal portion of the enzyme (putative transmembrane sequences): a putative pleckstrin homology domain and a distinct catalytic domain (3, 4). Because of the limited characterization of this isozyme, it will not be discussed further here.

View larger version (48K): [in this window] [in a new window]   Figure 1. Schematic sequence of PKC isozymes indicating the domain structure of the PKC subfamilies. The figure is a modification of a figure from Nishizuka (2).

The various PKC isozymes clearly mediate unique functions; even the ~50 amino acid difference in the alternatively spliced forms of ßPKC (ßI and ßIIPKC) appears to be responsible for the unique role of each ßPKC isozyme (5). A recent review in The FASEB J. examined potential differences between isozymes with respect to substrate specificity, phorbol ester binding, and sensitivity to synthetic inhibitors (6). This review focuses on the cPKC and nPKC subfamilies, their selective interactions with binding proteins that anchor them to different subcellular sites, and the functional consequences of these interactions.

Inactive PKC isozymes were thought to be present mainly in the cytosol, whereas their activators are hydrophobic and are present in the membrane. Centrifugation studies of the subcellular distribution of PKC isozymes demonstrated that agonists increased the amounts of PKC kinase activity (7) or immunoreactivity (8) in a 100,000 g pellet with corresponding decreases in the supernatant. Activated PKC was found to be associated with the plasma membrane, cytoskeletal elements, nuclei, and other subcellular components. Studies using conventional or confocal microscopy reveal a more complex and specific localization of PKC isozymes. Several isozymes are present within a single cell. Most inactive isozymes are localized to subcellular structures and, upon activation, translocate to new distinct intracellular sites. For example, PKC is localized to focal contacts in nonstimulated REF52 fibroblasts and translocates to the perinucleus after activation (9). ßIIPKC associates with fibrillar structures in nonstimulated cardiac myocytes and translocates to the perinucleus and cell periphery on activation (10, 11). In the same cells, PKC is localized to the nucleus and perinucleus before stimulation and translocates to cross-striated structures (possibly the contractile elements) and cell–cell contact regions after activation. Similar localization to cross-striated structures and cell–cell contact regions is observed when exogenous activated PKC is added to fixed cells (10, 11). The localization of overexpressed inactive PKC, but not other isozymes, to the Golgi apparatus in NIH 3T3 cells has also been demonstrated (12).

To illustrate these observations, Fig. 2 demonstrates the distinct localization of PKC in NG108–15 neuoroblastoma x glioma hybrid cells. In control cells (cultured in the absence of serum), PKC is localized to the Golgi area (as indicated by colocalization of PKC with the Golgi marker, BODIPY TR ceramide; data not shown). After activation by ß-phorbol 12-myristate 13-acetate (PMA; 100 nm) for 10 min, PKC is found in the perinucleus and nucleus ( Fig. 2) (13). In a minority of the cells, diffuse cytosolic staining is also observed ( Fig. 2). Taken together, these data suggest that both inactive and active PKC isozymes are localized to specific intracellular sites due to their binding to specific anchoring molecules.

View larger version (90K): [in this window] [in a new window]   Figure 2. PMA induces translocation of protein kinase C (PKC) from Golgi-like structures to the perinucleus and nucleus (13). NG108–15 cells were incubated with 100 nM PMA for 10 min. Cells were fixed and stained for PKC using isozyme-specific antibodies and fluorescein-labeled secondary antibodies. Slides were then scanned using a Bio-Rad 1024 confocal microscope. The pseudo-color image scale is displayed on the right of the images. In control cells, PKC is localized to the Golgi area (colocalization with the Golgi marker BODIPY TR ceramide). When PKC is activated by ß-PMA, it translocates to the perinucleus and nucleus.

The anchoring proteins for activated PKC isozymes were termed receptors for activated C-kinase [RACKs (14)]. We also suggest that there is another set of proteins that anchor inactive PKC isozymes. For example, there are likely PKC-specific binding proteins that anchor inactive PKC at the Golgi structures of NG108–15 cells. We refer to these proteins as receptors for inactive C-kinase isozymes, or RICKs. Each isozyme may have several different proteins that anchor it to different subcellular sites in the inactive state (RICKs). Similarly, there might be several RACKs that anchor PKC in the activated states (see subsequent text for supporting data). It is likely that the unique cellular functions of PKCs are determined by the binding of isozymes to specific anchoring molecules in close proximity to particular subsets of substrates and away from others.

	IDENTIFICATION OF ANCHORING PROTEINS

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 
An early observation that trypsin treatment of membranes inhibits the stable interaction of activated PKC with membranes (15) suggested that membrane proteins may bind activated PKC. Using an overlay assay, Wolf and Sahyoun (16) demonstrated that PKC binds to several proteins in the cell particulate fraction. They identified 110–115 kDa proteins from the cell cytoskeleton that bind PKC in a PS-dependent manner. These proteins were not further characterized. However, similar techniques were used by others to identify additional proteins that bind PKC, some of which are described below.

PS binding proteins/substrates
Using the overlay technique, as well as screening of an expression library, Jaken and collaborators (17) have identified additional PKC binding proteins. These proteins are all substrates of PKC and bind PS directly; a PS bridge between the binding proteins and PKC has been suggested to mediate this binding (17). These PKC binding proteins include talin and vinculin, myristoylated alanine-rich C-kinase substrate, a ß-adducin homolog, AKAP79 (18), clone 72 (19), and a related gene product, gravin/AKAP250 (20). Because activation of PKC is dependent on PS and DG (as well as on calcium for some of the isozymes), the finding that PS alone is sufficient for binding of PKC to these binding proteins (17) suggests that full activation of PKC is not required. Furthermore, these investigators found that PKC, for example, localizes to focal contact structures, where talin and vinculin are found in nonstimulated fibroblasts and in cultured renal cells (9, 21), and that activation by phorbol esters results in PKC translocation away from these proteins, as shown by cell fractionation (21).

Additional PKC binding proteins that bind PKC via a PS or lipid bridge and are substrates of PKC were cloned by interactive cloning that used autophosphorylated recombinant PKC (22). One protein (sdr-related gene product that binds to C-kinase, SBRC) is a substrate of PKC that binds to the regulatory domain of the enzyme in a PS-dependent manner and is phosphorylated in vivo after PKC activation by PMA (22). In vitro, SBRC does not appear to be an exclusive PKC binding protein; it also binds well to PKC, but not to PKC (22). Although PKC is activated in order to undergo autophosphorylation, it is possible that spontaneous inactivation occurs due to the prolonged incubation. Therefore, the binding proteins identified by this protocol may bind inactive PKC. Bruton tyrosine kinase (Btk) also binds to all the PKC isozymes in a lipid-dependent manner; in vitro studies demonstrate inhibition of this binding by PMA (23), suggesting that Btk binds inactive, or at least not fully activated, PKC.

Taken together, these data suggest that the above PS binding proteins/substrates could be anchoring proteins for inactive PKC. However, it has not yet been determined whether these proteins fulfill the criteria for RICKs. We predict that RICKs are proteins that bind PKC in an isozyme-specific and saturable manner, because individual PKC isozymes are differentially localized within each cell in the inactive state (e.g., ref 11). RICKs need not be substrates of PKC. However, they should demonstrate preferred binding of inactive PKC; PMA or other PKC activators should induce the release of PKC from these proteins. In this respect, characteristics of the putative RICKs are similar to those of the anchoring proteins for cAMP-dependent protein kinase (PKA), termed AKAPs. AKAPs bind PKA via its regulatory subunit, and activation of PKA after cAMP elevation induces detachment of the catalytic subunits and their translocation to new subcellular sites (20, 24).

Very recently, InaD has been identified as an anchoring protein for inactive PKC in the Drosophila eye, and is required for normal inactivation of a light stimulus in this organism (25). It appears that InaD anchors inactive eye PKC and is required for localization of the enzyme in close proximity to its site of action in the rhabdomeres. In the absence of InaD binding, eye PKC is subjected to proteolysis, suggesting that another role of anchoring PKC is to protect it from cellular proteases. The selectivity of InaD for eye PKC as compared to other PKC isozymes, and PMA- or activation-induced release of PKC from InaD, have not yet been determined. If these criteria are also met, InaD may be an eye PKC-selective RICK.

RACKs
Several proteins bind only activated PKC and fulfill the criteria for RACKs (14), proteins present in the cell particulate fraction that bind only activated PKC isozymes in a selective and saturable manner. A PS bridge should not be sufficient for binding of PKC to these RACKs (14); there should be direct protein–protein interaction (24, 26). Moreover, PKC binding to RACKs should not be inhibited by a substrate peptide (14), indicating that anchorage does not reflect binding of the catalytic site on PKC to a phosphorylation site on these proteins.

RACK1, a 36 kDa protein identified by screening a rat brain expression library for proteins that bind activated PKC, fulfills all the criteria for a RACK (14). RACK1 is not a substrate for PKC; however, in its presence, substrate phosphorylation by PKC is increased several-fold (27), suggesting that the PKC-RACK1 complex may be the active form of the enzyme in vivo (14, 27). RACK1 is colocalized with activated ßIIPKC to the perinucleus in cardiac myocytes (28), suggesting that it is the ßIIPKC-specific RACK.

An PKC-selective RACK has also been identified by expression cloning (29) using a fragment of PKC that contains the RACK binding site (see subsequent text). It also fulfills all the criteria for a RACK; similar to RACK1, it contains seven repeats of the WD 40 motif, first identified in the ß subunit of G-proteins (30, 31). It selectively binds PKC, but is not a substrate of any PKC. It is colocalized only with activated PKC (and not other isozymes) to cross-striated structures, perinucleus, and cell–cell contacts in cardiac myocytes (29). These data indicate that this RACK binds selectively activated PKC in intact cells. Because activated PKC isozymes are localized to several subcellular sites within the same cell (11), it is possible there is more than one RACK for each isozyme within the same cells. It is also possible that there are tissue-specific RACKs that allow for unique functions of individual isozymes in specific tissues.

Using the two-hybrid cloning system, Olson and collaborators (32) have identified an PKC substrate, termed PICK1 (protein interacting with C-kinase), which binds the catalytic fragment of PKC (amino acids 302–672). Most of PICK1 is in the perinucleus, where PKC is localized only after activation-induced translocation. Therefore, it appears that PICK1 best fits the category of RACKs, although determination of whether all the criteria for RACKs are met awaits further experiments.

Prekeris et al. (33) found that PKC, and not other PKC isozymes, binds to filamentous actin (F-actin) in vitro and in synaptosomes. Because only the activated form of PKC binds, F-actin appears to have the characteristics of an RACK (33). Blobe and collaborators (34) have found that, in vivo, in three different cell lines, F-actin also binds ßIIPKC but not ßIPKC. Binding to F-actin results in stimulation of ßIIPKC activity, similar to the effect of RACK1 (27). In vitro, ßIIPKC displays altered substrate specificity when bound to F-actin (34). These data suggest that F-actin may also have ßIIRACK characteristics. Whether both and ßIIPKC bind to F-actin in the same cells and whether their binding is competitive remain to be determined.

	MODEL OF PKC BINDING TO ANCHORING PROTEINS

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 
Figure 3 presents a model for PKC binding to its anchoring proteins. Inactive PKC is depicted as a folded rod with the pseudosubstrate autoinhibitory sequence at the amino terminus associated with the substrate site in the catalytic domain (35). In inactive PKC, a binding site for RICKs is exposed. In the presence of PKC activators, the rod unfolds and the RACK binding site becomes exposed, resulting in binding of PKC to its RACK. RACK-bound PKC is shown with the substrate binding site exposed and the RICK binding site unavailable for binding. Although direct experiments have not examined the ability of RICKs and RACKs to associate with PKC concomitantly, the immunofluoresence data and the inhibition of binding to at least some putative RICKs in the presence of PMA (22, 23) suggest exclusive binding of the enzyme either to RICKs or to RACKs. The scheme also reflects the findings that although there is a PS bridge between RACK and PKC (14), there is also direct protein–protein interaction (see below). Finally, there are no experimental data addressing a route for PKC translocation. For simplicity, the scheme illustrates a direct translocation of PKC between RICKs and RACKs ( Fig. 3).

View larger version (32K): [in this window] [in a new window]   Figure 3. A model of interaction of PKC with anchoring proteins. RICKs (in light green) bind inactive PKC whereas RACKs (in light blue) bind activated PKC. Although there is a PS bridge between the anchoring proteins and PKC, there is likely a direct protein–protein interaction as well. The trigger for and mechanism of shuttling PKC between RICKs and RACKs is not known (see text). The binding sites for RICKs and RACKs are depicted in the regulatory domain of PKC. However, additional binding sites for these anchoring molecules within the catalytic domain are likely to be present. Based on the differential immunolocalization of active and inactive PKC isozymes at different subcellular sites, the variable domains as well as unique sequence within the common domains are also expected to contribute to binding of PKC to these anchoring proteins.

	FUNCTIONAL ROLE OF ANCHORING PROTEINS

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 
If translocation of PKC from RICKs to RACKs is required for the enzyme to function, identification of the domains on RICKs, RACKs, and their respective PKC isozymes that mediate binding should allow direct examination of the functional role of these anchoring proteins. Fragments and peptides derived from PKC that contain the corresponding RACK and RICK binding sites could be used in intact cells to compete with anchoring of PKC and, thus, alter its function.

Identification of domains in PKC that mediate binding to anchoring molecules
C1 domain
Studies from Anderson's laboratory (12) indicate an isozyme-selective interaction with the Golgi apparatus that is mediated by the C1 domain of PKC and is independent of the pseudosubstrate sequence. Overexpressed inactive PKC and a fragment (amino acids 166–297) containing the PKC C1 domain are both localized to the Golgi apparatus in nonstimulated cells. Anchoring to Golgi is probably via a RICK, because activation by PMA results in translocation of the enzyme or fragment away from this location (36). Therefore, subcellular localization and binding of inactive PKC to its RICK in these cells appear to be mediated, in part, by regions in the C1 domain that are exposed in inactive PKC ( Fig. 3).

A recent study by Kawakami and collaborators (23) demonstrates that the C1 domains of conventional, novel, and atypical PKC isozymes were all found to bind the pleckstrin homology domain of Btk of the Tec family in the inactive form. As discussed above, the interaction of the PKC isozymes with Btk is inhibited by PMA, indicating Btk is likely a RICK. Phorbol ester exerts an inhibitory effect on the in vitro interaction only when classical and novel PKC isozymes are used; as expected, PMA does not inhibit binding of the PMA-insensitive isozyme PKC to Btk (23). Therefore, the C1 region is likely to contain a RICK binding site whose conformation is sensitive to phorbol ester binding to C1.

The binding site for some of the PKC/PS binding proteins identified by Jaken and Jones (37) has also been mapped to the C1 domain and the pseudosubstrate sequence within it [PKC (amino acids 19–31)]. A peptide corresponding to the pseudosubstrate sequence binds to these PKC binding proteins in a PS-dependent manner, and an PKC mutant lacking the pseudosubstrate sequence shows diminished binding to these proteins in vitro as compared to wild-type PKC (38).

Finally, an isozyme-unique sequence within the C1 domain of PKC (amino acids 223–228) mediates the binding of this activated isozyme to F-actin (33). F actin appears to be a RACK because it binds only activated PKC (33). In contrast, the proteins described above bind inactive PKC, and activation causes their dissociation from the binding proteins; hence, these binding proteins appear to be RICKs. Yet both types of proteins bind PKC via the C1 region. Taken together, the data suggest that both RACK and RICK binding sites may reside partly within the C1 domain, suggesting that there are at least two protein–protein interaction sites within this phorbol ester binding domain. In this respect, a C1-like domain in Raf kinase is also involved in protein–protein interaction with the small G-protein, Ras (39).

C2 domain
The C2 domain also mediates the PS bridge between PKC and some of its binding proteins that appear to be RICKs (38). However, no direct and isozyme-specific protein–protein interactions have yet been demonstrated between the C2 domain and these binding proteins. In contrast, earlier studies demonstrated that the C2 domain of PKC mediates at least some of the direct protein–protein interaction between PKC and RACKs (40). The RACK1 binding site, located in the C2 domain of the C2-containing cPKCs, was further mapped to amino acids 186–198, 209–216, and 217–226 (28). X-ray and nuclear magnetic resonance studies of the C2 domains suggest that sequences corresponding to the RACK1 binding sites in PKC are on three exposed ß-strands in the domain (41). Furthermore, peptides corresponding to these three ß-strands specifically inhibit activation-induced translocation of the C2-containing cPKC isozymes and not the C2-less nPKCs (28). Therefore, RACK1-ßPKC interactions are mediated by multiple protein–protein contacts occurring at the interface between these molecules. Because each cPKC isozyme is differentially localized within the same cell (11), unique sequences within the C2 domain or elsewhere in PKC must also be part of the RACK1 binding site.

Variable domains
Recent studies indicate that binding of the C2-less nPKC isozymes to their RACKs is via the V1 domain, which has some homology to the C2 domain of the cPKCs (42). Fragments containing the V1 domain of PKC or PKC (amino acids 1–142) selectively compete with the corresponding endogenous enzymes and specifically prevent their translocation after cell stimulation by phorbol ester or norepinephrine without affecting the translocation of each other or cPKCs (43). The RACK binding site on PKC has been further mapped to amino acids 14–21; a peptide corresponding to this sequence selectively inhibits translocation of PKC, but not translocation of other isozymes (43).

It is likely that other variable domains also contribute to isozyme-specific anchoring and function. For example, the V5 domain of ßI and ßIIPKC must contribute to their distinctive localizations because it is the only domain that differs between the two isozymes. Indeed, a sequence within the V5 domain of ßIIPKC (amino acids 629–656 and 667–673) determines its selective binding to F-actin (34). Moreover, this domain contains autophosphorylation sites (44, 45), and the extent of autophosphorylation alters 1) ßIIPKC localization to the cell particulate fraction (46), 2) catalytic activity, and 3) cellular function of this isozyme (5).

Modulation of PKC function by peptides derived from the RACK binding site
Inhibitors
If anchoring is required for the proper function of individual PKC isozymes (see above), then inhibition of anchoring should alter function. This prediction has been tested only for the anchoring of activated PKC isozymes to RACKs. Peptides that mimic either the PKC binding site on RACKs (see ref 24 for review) or the RACK binding site on PKC (see below) are translocation inhibitors of PKC that inhibit the function of the enzyme in vivo. Introduction of peptides corresponding to the RACK1 binding site on the C2 domain of cPKC into Xenopus oocytes inhibits ßPKC translocation and oocyte maturation, a ßPKC-regulated function in these cells (28). The same peptides inhibited phorbol ester-dependent regulation of L-type calcium channel activity in cardiac myocytes (47). In these cells, stimulation of ß1-adrenergic receptors by isoproterenol increases L-type calcium activity and PMA suppresses this effect by ~50%. When the recording electrode contained 100 nM of two ßC2-derived peptides (ßC2–2 and ßC2–4), PMA-induced inhibition was almost completely abolished. Therefore, a cPKC isozyme, possibly ßIIPKC, mediates PMA-induced regulation of this channel (47).

The PKC V1 fragment containing the RACK binding site on PKC, and an eight amino acid peptide derived from it [PKC (14–21)], block activation-induced translocation of PKC in cardiac myocytes. Both the V1 fragment and the peptide specifically inhibit phorbol ester- or norepinephrine-induced inhibition of contraction in cardiac myocytes (43). In contrast, the corresponding PKC V1 fragment that specifically inhibits phorbol ester-induced translocation of PKC or the C2-derived peptides corresponding to the RACK binding site in the cPKCs do not modulate contraction rate (43). The above isozyme-selective inhibitors were also used to demonstrate the role of PKC in modulating neuronal growth factor responses in PC12 cells (48) and glucose-induced insulin secretion from ß-islet cells (49). These data demonstrate that the use of isozyme-selective translocation inhibitors allows us to determine for the first time the function of individual PKC isozymes in intact cells.

Activators
The ability of PKC translocation inhibitors to selectively inhibit the function of individual isozymes indicates that translocation is required for PKC function. Therefore, translocation activators should be agonists of PKC function, independent of the amount of second messengers that normally activate PKC. Based on the model presented in Fig. 3, a peptide that binds PKC—causing detachment from RICKs, exposure of the catalytic site, and enabling anchoring to RACKs—should be a PKC agonist. Two peptides with agonist activity have been identified so far; both peptides mimic insulin-induced Xenopus oocyte maturation in the absence of insulin (50, 51). When introduced into cells, they cause cPKC translocation and activate a ßPKC function (50, 51). As with translocation inhibitors, it is likely that isozyme-selective translocation activators will be identified.

	OPEN QUESTIONS

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 
It appears that the localization and function of PKC isozymes are determined by binding to anchoring proteins at specific subcellular sites. Several PKC binding proteins have been proposed to anchor PKC isozymes to these sites. The details of the complex PKC-mediated signaling requiring multiple protein–protein interactions are only beginning to be uncovered. Progress has been made in identifying PKC translocation inhibitors and activators based on PKC-RACK interactions. However, many questions remain to be answered. Does activation of PKC result in shuttling of the isozymes between RICKs and RACKs; if so, what is the molecular mechanism underlying this shuttling? How does elevation of DG levels trigger PKC translocation, and where does PKC encounter this second messenger? What are the binding sites for RACKs and RICKs on each isozyme? What is the role of PS and other lipids or lipid metabolites in anchoring? An organic soluble factor (presumably a lipid) provides an isozyme-specific activator required for lamin B kinase activity of ßIIPKC (52). How do these lipid factors affect PKC localization and do they correspond to RACK-mediated localization? Do RACKs concomitantly bind PKC and its substrates? Could other proteins bind to the same anchoring molecules to create signaling complexes? Such signaling complexes have been identified. For example, recent studies show that PKC is anchored to a PKA anchoring protein, AKAP79 (18, 20); and PKC is anchored with phospholipase Cß and an ion channel to InaD, a scaffolding protein in the Drosophila eye (25). In addition, mammalian phospholipase C binds to RACKs (40). 14–3-3 may be an additional example of an anchoring protein for multiple signaling proteins; it binds and regulates the activity of PKC, Raf, and several other signaling proteins (53). These data suggest that signaling proteins may be organized in a multienzyme complex that might promote cross-talk between diverse signaling mechanisms.

As discussed above, altered PKC localization affects function, presumably because of altered accessibility of the kinase to protein substrates. Another tier of regulation of PKC signaling and a potential cross-talk between PKC and other signal transduction events could be modification of the anchoring proteins. Indeed, we find that the localization pattern of activated PKC isozymes can be dependent on the type of stimulus (54, 55). For example, localization of the (but not PKC) isozyme is different after transforming growth factor ß1 treatment as compared to PMA or norepinephrine treatment of the cardiac myocytes cells (11, 54). These data suggest that other factors may alter RACK localization or accessibility for binding by the activated isozyme. Finally, recent studies demonstrate a role for phosphorylation of PKC in the regulation of PKC binding to the cytoskeleton (56). This phosphorylation may regulate shuttling of PKC isozymes from one subcellular site to another or may increase the affinity for a subset of anchoring proteins (RICKs or RACKs) to alter PKC function.

The potential therapeutic value of isozyme-selective translocation inhibitors and activators is likely to be of great interest to both basic research and the pharmaceutical industry. PKC fragments and peptides that mimic the binding sites for their isozyme-specific RACKs can be directly introduced into cells to determine the role of individual isozymes in cell functions. Furthermore, these fragments and peptides can be used in high throughput assays to identify new drugs with isozyme-selective inhibitor or activator activity.

	ACKNOWLEDGMENTS

 
The work from D.M.R.'s laboratory was supported in part by The National Institutes of Health (NIH) (RO1 HL-43380, RO1 HL-52141) and The American Cancer Society (BE-158). A.S.G. was supported in part by NIH grants RO1 10039 and RO1 AA10030. D.M.R. thanks all the members of her laboratory for helpful discussions.

	FOOTNOTES

 
¹ Correspondence: Department of Molecular Pharmacology, Stanford University, School of Medicine, Stanford, CA 94305–5332, USA. E-mail: mochly{at}leland.stanford.edu

² Abbreviations: PKC, protein kinase C; PS, phosphatidylserine; DG, diacylglycerol; PMA, ß-phorbol 12-myristate 13-acetate; RACKs, receptors for activated C-kinase; RICKs, receptors for inactive C-kinase isozymes; Btk, Bruton tyrosine kinase; SBRC, sdr-related gene product that binds to C-kinase; V, variable; PICK1, protein interacting with C-kinase; AKAP, A-kinase anchoring protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION IDENTIFICATION OF ANCHORING... MODEL OF PKC BINDING... FUNCTIONAL ROLE OF ANCHORING... OPEN QUESTIONS REFERENCES

 

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