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Mapping of a Molecular Determinant for Protein Kinase C βII Isozyme Function*

In human erythroleukemia (K562) cells, the highly related protein kinase C (PKC) α and PKC βIIisozymes serve distinct functions in cellular differentiation and proliferation, respectively. Previous studies using two domain switch PKC chimera revealed that the catalytic domains of PKC α and βII contain molecular determinants important for isozyme-specific function (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9156–9160). We have now analyzed a panel of PKC chimeras to determine the specific region within the catalytic domain important for PKC βII function. A cellular assay for PKC βII function was devised based on the finding that PKC βII selectively translocates to the nucleus and phosphorylates nuclear lamin B in response to the PKC activator bryostatin. This response is strictly dependent upon expression of PKC βII or a PKC chimera that functions like PKC βII. We demonstrate that a PKC α/βIIchimera containing only the carboxyl-terminal 13 amino acids from PKC βII (βII V5) is capable of nuclear translocation and lamin B phosphorylation. These results are consistent with our recent observation that the PKC βII V5 region binds to phosphatidylglycerol (PG), a potent and selective PKC βII activator present in the nuclear membrane (Murray, N. R., and Fields, A. P. (1998) J. Biol. Chem. 273, 11514–11520). Soluble βII V5 peptide selectively inhibits PG-stimulated PKC βII activity in a dose-dependent fashion, indicating that PG-mediated activation of PKC βII involves interactions with the βII V5 region of the enzyme. We conclude that βII V5 is a major determinant for PKC βIInuclear function and suggest a model in which binding of PG to the βII V5 region stimulates nuclear PKC βIIactivity during G2 phase of the cell cycle.

Protein kinase C (PKC) 1 is a family of serine/threonine kinases that play crucial roles in various signaling processes, including cellular proliferation and differentiation (1)(2)(3)(4). Molecular cloning studies have shown that the PKC family has at least 12 distinct members classified into three groups according to their structure, calcium requirement, and cofactor dependence (5,6). The fact that PKC isotypes exhibit different patterns of tissue expression, subcellular localization, and activator/substrate specificity implies that there is functional diversity among isotypes (6 -10). Previous studies demonstrated that PKC ␣ and ␤ II are involved in the differentiation and proliferation of a variety of cell types (3,10). In human erythroleukemia (K562) cells, we determined that PKC ␣ is involved in PMA-induced cytostasis and megakaryocytic differentiation (10). PKC ␤ II , on the other hand, is required for K562 cell proliferation (10). PKC ␤ II selectively translocates to the nucleus during the G 2 /M phase transition of cell cycle and leads to direct phosphorylation of the nuclear envelope polypeptide lamin B at mitosis-specific sites involved in mitotic nuclear lamina disassembly (9,(11)(12)(13)(14)(15).
In order to study the molecular basis of PKC ␣ and ␤ II isozyme function, we expressed two domain switch chimeras between the regulatory and catalytic domains of PKC ␣ and ␤ II in K562 cells (16). These chimeras demonstrated that the catalytic domains of PKC ␣ and ␤ II contain determinants that are critical for isozyme-specific function (16). Specifically, a PKC ␤ II /␣ chimera, consisting of the regulatory domain of PKC ␤ II and the catalytic domain of PKC ␣, exhibited a phenotype resembling wild type PKC ␣ (16). Conversely, a PKC ␣/␤ II chimera, composed of the regulatory domain of PKC ␣ and the catalytic domain of PKC ␤ II , behaved like wild type PKC ␤ II (16).
Based upon these findings, we wished to define the isozymespecific determinants within the catalytic domain of PKC ␤ II . For this purpose, we constructed and expressed a series of ␣/␤ II PKC chimeras in which the variable and constant regions within the catalytic domain of PKC ␤ II were replaced by the corresponding PKC ␣ regions. Our results demonstrate that the V5 region of PKC ␤ II (␤ II V5), consisting of the carboxylterminal 13 amino acids of PKC ␤ II , contains the molecular determinant necessary for nuclear translocation and activation of the enzyme. These results are interesting, in light of our recent studies demonstrating that the V5 region of PKC ␤ II binds phosphatidylglycerol (PG), a nuclear activator of the enzyme (17). A soluble peptide corresponding to ␤ II V5 inhibits PG-stimulated PKC ␤ II activity, indicating that interactions between PG and the ␤ II V5 region are important for PG-mediated activation of PKC ␤ II . Based on these observations, we propose a model for the cell cycle-regulated activation of nuclear PKC ␤ II . PKC chimeras were constructed by two-step polymerase chain reaction (PCR) as described previously (16). The first step in the chimera construction was to amplify the desired PKC ␣ and PKC ␤ portions of the chimera using the appropriate 5Ј and 3Ј end primers, internal chimeric primers, and either PKC ␣ or PKC ␤ II cDNA as a template. The internal primers were constructed so that they would anneal in the second PCR step. Products from the first PCR step were gel-purified and combined, along with 5Ј and 3Ј end primers, and the complete chimera produced by extension and amplification. The primers used for construction of the chimeras are presented in Table I. The end primers contained restriction sites (5Ј KpnI and 3Ј NheI) to facilitate subsequent cloning. Completed chimeras were gel-purified and cloned into the TA PCR cloning vector pCR 2.1 (Invitrogen). The chimeras were restricted from pCR 2.1 using KpnI and NheI and ligated into the KpnI and NheI sites within the multiple cloning site of the episomal expression vector pREP4 (Invitrogen).
Transfection and Expression of PKC Chimeras in K562 Cells-Human erythroleukemia K562 cells (ATCC) were maintained in suspension culture as described previously (10). Cells were transfected with the pREP4 plasmids containing the PKC chimera constructs using DOTAP lipofection reagent (Boehringer Mannheim) following the manufacturer's protocol. 24 h after transfection, fresh medium containing 250 units/ml hygromycin B (Calbiochem) was added to the cultures and transfectants selected for 3-4 weeks. K562 cell transfectants were maintained continuously in growth medium supplemented with 250 units/ml hygromycin B. Expression of PKC chimeras was determined by immunoblot analysis using previously characterized isotype-specific antibodies directed against the V5 regions of PKC ␣ and ␤ II (14). For immunoblotting, K562 cell transfectants were washed with cold phosphate-buffered saline, sonicated for 30 s in SDS sample buffer (18), and boiled for 5 min. Total cell extracts from 1 ϫ 10 5 cells were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Schleicher & Schuell), and subjected to immunoblot analysis as described previously (10). The chimeric nature of the chimeras was confirmed by immunoblot analysis using isozyme-specific antibodies against the V3 region of PKC ␣ and ␤ II as described previously (16).
Drug Treatment, Isolation of Nuclear Envelopes, and Lamin B Phosphorylation-K562 cell transfectants carrying either control vector (pREP4) or PKC chimera-containing pREP4 vectors were treated with 30 nM PMA (LC Laboratories) or diluent (0.1% Me 2 SO). Previous studies demonstrated that this PMA treatment leads to loss of immunologically detectable PKC ␤ II and loss of bryostatin-mediated lamin B phosphorylation (10,16). Following the 48-h PMA treatment, total cell lysates were prepared from an aliquot of cells and subjected to immunoblot analysis using isotype-specific PKC ␣ V5 and ␤ II V5 antibodies as described above. The remainder of the cells were incubated in the presence and absence of bryostatin 1 (100 nM, LC Laboratories) for 30 min at 37°C, followed by nuclear envelope isolation and determination of lamin B phosphorylation as described previously (16).
Assay of Protein Kinase C ␤ II Activity in Vitro-In vitro protein kinase C assays were carried out using purified baculovirus-expressed human PKC ␤ II as described previously (15). Briefly, reactions were performed in assay buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM In most reactions, 250 g/ml (315 M) dioleoyl PG (Avanti Polar Lipids) was added to maximally stimulate PKC ␤ II activity (17). Synthetic peptides corresponding to ␤ II V5 (CFVNSEFLKPEVKS) or ␣ V5 (CQFVHPILQSSV) were added at the concentrations indicated in the figure legends. Histone H1 phosphorylation was quantitated using phosphorimaging analysis as described previously (12).

Construction and Expression of PKC Chimeras in K562
Cells-Based on our previous finding that the catalytic domain of PKC ␤ II is crucial for PKC ␤ II function (16), we generated a series of PKC chimeras in which the variable and constant regions within the catalytic domain of PKC ␤ II were exchanged with the corresponding sequences from PKC ␣ by two-step PCR as depicted in Fig. 1. To investigate the biochemical properties of these chimeras in intact K562 cells, chimeric PKC constructs were subcloned into the pREP4 episomal expression vector and transfected into K562 cells. Expression of the PKC chimeras was confirmed by immunoblot analysis with antibodies against the carboxyl-terminal V5 regions of PKC ␣ and ␤ II (Fig. 2). Expression of each of the chimeras containing the ␤ II V5 region (␤ II V4, ␤ II C4, ␤ II V5, ␤ II 1504, and ␤ II 1851) was confirmed using the PKC ␤ II V5 antibody ( Fig. 2A). Expression of the ␣ ␤ II ␣ chimera, which contains the carboxyl-terminal V5 region from PKC ␣, was confirmed using the PKC ␣ V5 antibody (Fig.  2B). In each case, a band with apparent molecular mass of 85 kDa was detected, corresponding in size to intact PKC ␣ and ␤ II protein. The level of expression for all chimeras was determined by densitometric analysis to be between 2-and 3-fold that of the endogenous levels of PKC ␣ or PKC ␤ II . The chimeric nature of each of the chimeras was confirmed by immunoblot analysis using the appropriate PKC ␣ and ␤ II -specific V3 antibodies as described previously (Ref. 16; data not shown).
PKC Chimera Expression Persists after PMA Treatment-Our previous studies demonstrated that treatment of K562 cells with PMA for 48 h leads to an increase in PKC ␣ expression and a loss of PKC ␤ II expression (10). We therefore wished to assess the fate of PKC chimeras expressed in K562 cells following treatment with PMA. For this purpose, K562 cell transfectants were treated with PMA (30 nM) for 48 h. Transfectants were harvested and immunoblotted as described under "Experimental Procedures" (Fig. 3). As expected, endogenous PKC ␤ II expression was dramatically reduced as a consequence

5Ј-CCGCGCTAGCTTAGCTCTTGACTTCGG-3Ј
Internal chimeric primers a ␤ II V4 AS of PMA treatment (Fig. 3A, compare lanes 1 and 2) (10,16). In contrast, the levels of PKC ␣ were increased as previously reported (10, 16) (Fig. 3B, compare lanes 1 and 2). Interestingly, in contrast to endogenous PKC ␤ II , the expression of each of the transfected PKC chimeras (␤ II V4, ␤ II C4, ␤ II 1504, ␤ II 1851, ␤ II V5, and ␣␤ II ␣) persists after PMA treatment (Fig. 3, A  and B). Ability of PKC Chimeras to Reconstitute PKC ␤ II Function-The observation that expression of PKC chimera persists after chronic PMA treatment while endogenous PKC ␤ II levels are dramatically reduced suggested a strategy to specifically determine whether the transfected PKC chimeras could function like PKC ␤ II in intact K562 cells without the contribution of endogenous PKC ␤ II . This strategy is predicated upon several key observations. First, PKC ␤ II is selectively translocated and activated at the cell nucleus in response to bryostatin but not PMA (9,11,14). Second, bryostatin-mediated translocation of PKC ␤ II to the nucleus leads to direct phosphorylation of the nuclear envelope polypeptide lamin B (11)(12)(13)(14)(15). Third, the ability of bryostatin to stimulate nuclear lamin B phosphorylation is strictly dependent upon the expression of PKC ␤ II (16). Thus, when K562 cells are treated with PMA, both PKC ␤ II expression and bryostatin-mediated lamin B phosphorylation is lost (16). Therefore, we assessed the ability of the transfected PKC chimeras to reconstitute bryostatin-mediated lamin B phosphorylation in PMA-treated K562 cell transfectants as described under "Experimental Procedures" (Fig. 4). Treatment of K562 cells containing an empty control vector with PMA leads to the expected loss in bryostatin-mediated lamin B phosphorylation, whereas cells not treated with PMA exhibited robust lamin B phosphorylation in response to bryostatin (Fig. 4, compare panels labeled Control (ϪPMA) and Control (ϩPMA). K562 cells expressing the ␤ II V4, ␤ II C4, ␤ II 1504, ␤ II 1851, and ␤ II V5 chimeras were all capable of reconstituting bryostatin-mediated lamin B phosphorylation after treatment with PMA. In contrast, cells expressing the ␣␤ II ␣ chimera were incapable of reconstituting the bryostatin-mediated response. From these results, we conclude that chimeras containing the functional determinant important for PKC ␤ II function are capable of reconstituting PKC ␤ II -dependent lamin B phosphorylation. Furthermore, our analysis localizes this functional determinant to the extreme carboxyl-terminal 13 amino acids of PKC ␤ II (␤ II V5), since a chimera containing only these 13 amino acid residues from PKC ␤ II (PKC ␤ II V5) is capable of functioning in this PKC ␤ II -selective pathway.
PG-stimulated PKC ␤ II Activity Involves Interactions with the V5 Region of PKC ␤ II -The results from the PKC chimera studies demonstrate that the carboxyl-terminal V5 region of PKC ␤ II represents a molecular determinant for PKC ␤ II -selective function in intact K562 cells. In recent studies, we identified a component of the nuclear membrane, termed nuclear membrane activation factor (NMAF), which selectively acti- vates PKC ␤ II (19). More recently, we identified NMAF as PG (17). PG was found to be a potent and selective activator of PKC ␤ II , stimulating the enzyme 3-5-fold above the level of activity seen in the presence of optimal concentrations of PS, DAG, and calcium (19). We also demonstrated that PG binds selectively and saturably to the carboxyl-terminal region of PKC ␤ II (17), suggesting that PG stimulates PKC ␤ II activity through interactions involving ␤ II V5. To specifically test this hypothesis, we assessed the effect of soluble ␤ II V5 peptide on PG-stimulated PKC ␤ II activity in vitro (Fig. 5). As reported previously (17) addition of 315 M PG stimulates PKC ␤ II more than 4-fold over the level in the absence of PG. This stimulation is specific for PG since it is not observed in the presence of 370 M PS (data not shown). Inclusion of ␤ II V5 peptide in the kinase assay leads to inhibition of PG-stimulated PKC ␤ II histone kinase activity (Fig. 5A, lane 5). Inhibition is specific for the ␤ II V5 peptide, since no inhibition is observed using the corresponding V5 peptide from PKC ␣ (Fig. 5A, lane 4). Inhibition by ␤ II V5 peptide is dose-dependent with an apparent IC 50 of ϳ100 M (Fig. 5B), whereas significant inhibition was not observed with the corresponding ␣ V5 peptide. Interestingly, the ␤ II V5 peptide inhibits only the PG-stimulated component of PKC ␤ II activity and not calcium-, DAG-, and PS-stimulated activity. These results are consistent with the conclusion that PG mediates activation through the carboxyl-terminal V5 region of PKC ␤ II , whereas calcium, DAG, and PS mediate activation by binding to the C1 and C2 regions within the regulatory domain of the enzyme. DISCUSSION The PKC family of serine/threonine kinases serves critical roles in cellular function. Recent studies have begun to elucidate specific roles for individual PKC isozymes (1,3,5). We have identified roles for the three major PKC isozymes in human K562 leukemia cells. K562 cells express PKC ␣, ␤ II , and , and each of these isozymes serve distinct functions in these cells. PKC ␣ is important in cellular differentiation (10), PKC ␤ II is required for cellular proliferation (10) and PKC plays a critical role in leukemia cell survival and resistance to apoptosis (20). However, despite our knowledge of the importance of these isozymes in cellular physiology, less is known about the specific pathways in which these isozymes function, the relevant cellular targets of their action, and the mechanisms by which they maintain isozyme identity within the intact cell. We have addressed these questions in the case of PKC ␤ II . PKC ␤ II is required for cell cycle progression through the G 2 /M phase transition (12,13). PKC ␤ II is activated at the nucleus during late G 2 phase just prior to mitosis in synchronized cells (12,13). At the nucleus, PKC ␤ II mediates direct phosphorylation of the nuclear envelope polypeptide lamin B on sites involved in mitotic nuclear lamina disassembly (12,13,15). Inhibition of PKC ␤ II activation leads to cell cycle arrest in G 2 phase, demonstrating the importance of nuclear PKC ␤ II activation and lamin B phosphorylation in entry into mitosis (13).
Nuclear PKC ␤ II activation is regulated by several factors. First, a nuclear phosphoinositide-specific phospholipase C activity generates a peak of diacylglycerol at the nuclear membrane during G 2 phase that serves to activate nuclear PKC ␤ II (21). Second, the nuclear membrane contains a potent activator of PKC ␤ II that stimulates its activity above the level observed in the presence of optimal calcium, DAG, and PS (17,19). This activator, originally termed NMAF, was recently identified as the phospholipid PG (17). The selectivity of PG for PKC ␤ II activation suggested that PG might function through interactions within the carboxyl-terminal V5 region of PKC ␤ II . Indeed, we have demonstrated that the carboxyl-terminal region of PKC ␤ II binds selectively and saturably to PG-containing vesicles, but not to vesicles containing other phospholipids (17).
In the present study, we wished to determine the molecular determinants on PKC ␤ II that allow it to translocate to the nucleus and phosphorylate lamin B in intact cells, a process that we have demonstrated requires PKC ␤ II expression (16). We previously demonstrated that a chimera between PKC ␣   FIG. 4. PKC chimeras can reconstitute lamin B phosphorylation. K562 cells transfected with either control vector or the indicated PKC chimeras were harvested following 48 h PMA treatment. After washing to remove the PMA, cells were incubated without or with bryostatin 1 (bryo, 100 nM) for 30 min and assayed for lamin B phosphorylation as described previously (16). The labels are the same as described in Fig. 3. Results are representative of three independent experiments.
FIG. 5. PG-stimulated PKC ␤ II activity is inhibited by the ␤ II V5 peptide in a dose-dependent manner. Recombinant human PKC ␤ II was incubated in kinase buffer in the absence or presence of the indicated concentration of either ␤ II V5 or ␣V5 peptide for 15 min at 25°C as described under "Experimental Procedures." Activity was monitored by incorporation of 32 P into purified histone H1 (10 g) as described previously (12). Panel A, autoradiograph of PKC ␤ II -mediated histone H1 phosphorylation by PKC ␤ II . Reactions were as follows. and PKC ␤ II containing the catalytic domain of PKC ␤ II was capable of translocating to the nucleus and phosphorylating lamin B (16). In contrast, a chimera containing the catalytic domain of PKC ␣ was not capable of mediating this response (16). From these data, we concluded that regions within the catalytic domain of PKC ␤ II serve as a molecular determinant of PKC ␤ II function (16). We have now used further PKC ␣/␤ II chimeras to map this molecular determinant to the extreme carboxyl-terminal 13 amino acids of PKC ␤ II . The results of this study are interesting in light of our recent finding that the V5 region of PKC ␤ II mediates binding to PG (17). In fact, we now demonstrate that a ␤ II V5 peptide selectively inhibits PG-stimulated PKC ␤ II activity in a dose-dependent fashion. These results indicate that interactions between PG and ␤ II V5 are important for PG-stimulated PKC ␤ II activity.
The identification of the carboxyl-terminal region of PKC ␤ II as an important region regulating isozyme specific function is consistent with recent studies suggesting the importance of this region to PKC function. The V5 region of PKC ␣ has recently been shown to interact with PICK 1, a PKC-binding protein, through PDZ domain-like interactions (22). PICK1 has been suggested to play a role in targeting PKC ␣ to appropriate intracellular sites where it can transduce isozyme-specific signals (22). Likewise, we have demonstrated that interaction of the V5 region of PKC ␤ II , in this case with the phospholipid PG, is important for the nuclear translocation and activation of the enzyme at the nuclear membrane, where it is required for nuclear lamina disassembly and entry into mitosis (17). Finally, studies investigating the differential regulation of PKC ␤ I and ␤ II activity by calcium have led to the suggestion that the V5 region of PKC ␤ II interacts with the C2 region of the enzyme to influence calcium-and PS-mediated enzyme activation (23).
Given our recent data on nuclear activation of PKC ␤ II , the proposed interdomain interactions involving the C2 and V5 regions of PKC ␤ II , and related studies on the mechanism of PKC membrane translocation and activation (24), we propose a working model for the cell cycle-regulated, nuclear activation of PKC ␤ II as illustrated in Fig. 6. Soluble PKC ␤ II is stably phosphorylated on at least three known sites (24,25). First, PKC ␤ II is phosphorylated by a putative protein kinase C kinase at threonine 500 in the activation loop of the enzyme (24,25). Phosphorylation at threonine 500 makes PKC ␤ II catalytically competent and triggers subsequent autophosphorylation of threonine 641 and serine 660 in the carboxyl-terminal region of the enzyme. Phosphorylation of these sites stabilizes the catalytically competent conformation of the enzyme and allows its release into the cytosol (24,25). In our model, a nuclear PI-PLC isozyme(s), whose activity is linked to cell cycle by an unknown mechanism, is activated during the G 2 phase of cell cycle (21). Activation of nuclear PI-PLC leads to generation of inositol trisphosphate (IP 3 ) and DAG. We propose that IP 3 binds to nuclear IP 3 receptors to mobilize intracellular calcium stores, leading to a rise in intracellular calcium concentrations. It has been demonstrated that nuclei contain functional IP 3 receptors (26) and that intracellular calcium concentrations rise in synchronized cells just prior to mitosis (27)(28)(29). Furthermore, inhibition of this rise in intracellular calcium, using calcium chelators such as BAPTA, leads to cell cycle arrest in G 2 phase, demonstrating the importance of calcium in entry into mitosis (29). We propose that one function of elevated calcium levels is to translocate PKC ␤ II to the inner nuclear membrane. We have not directly determined whether this step involves translocation of PKC ␤ II from cytosolic pools, or alternatively involves translocation of inactive PKC ␤ II from a nucleoplasmic pool. However, based on our fractionation studies (9,(11)(12)(13)(14)(15) and the immunofluorescence studies of others in cultured myocytes (30), we favor the hypothesis that this step represents translocation of cytosolic PKC ␤ II to the nucleus. As has been previously demonstrated, binding of calcium to PKC increases its membrane affinity leading to membrane translocation and an increase in affinity of the enzyme for PS (31)(32)(33). Membrane bound PKC coordinately binds multiple PS molecules, possibly within the C2 region of the enzyme (34,35). It has been demonstrated that PKC induces calcium-dependent clustering of acidic phospholipids into microdomains that may facilitate cooperative binding of phospholipid to the C2 domain (36). We propose that nuclear PG coclusters with PS during this phase of nuclear membrane binding. In the presence of DAG, PKC activation occurs, through displacement of the FIG. 6. A proposed model for the nuclear activation of PKC ␤ II . Prior to activation, PKC ␤ II is phosphorylated at multiple sites within the catalytic domain (indicated by P) giving rise to a catalytically competent enzyme that resides in the cytosol (Refs. 24 and 25; I). During G 2 phase of cell cycle, a nuclear PI-PLC is activated, leading to the generation of nuclear DAG (21) and IP 3 . IP 3 activates nuclear IP 3 receptors (26), leading to elevation of intranuclear calcium concentrations just prior to mitosis (27)(28)(29). In the presence of elevated calcium and acidic phospholipids, PKC interacts with the nuclear membrane (II). Membrane binding leads to clustering of acidic phospholipids including PS and PG to microdomains within the membrane (III). The binding of DAG to the C1 region promotes cooperative binding of PS to the C2 domain of PKC ␤ II , thereby inducing a conformational change that releases the pseudosubstrate domain of PKC ␤ II from the active site. The carboxyl-terminal region of the enzyme is brought into juxtaposition with the C2 domain via specific binding of ␤ II V5 to clustered PG, leading to full activation of the enzyme. Activated PKC ␤ II directly phosphorylates its nuclear substrate lamin B, leading to mitotic nuclear lamina disassembly and entry into mitosis (38). pseudosubstrate domain from the active site of the enzyme (37). It has recently been proposed that the V5 region of PKC ␤ II interacts with the C2 region of the enzyme when it is in its active conformation (23). We further propose that these V5-C2 interdomain interactions are mediated, at least in part, by binding of the V5 region to PG that is clustered in the same nuclear membrane microdomains containing the PS involved in membrane/C2 domain interactions. Once optimally activated at the inner nuclear membrane, PKC ␤ II directly phosphorylates lamin B at Ser-405 within its carboxyl-terminal globular domain (12,15). Lamin B is an ideal substrate for nuclear membrane-bound PKC ␤ II since it is intimately associated with the inner nuclear membrane surface by virtue of its carboxyl-terminal isoprenyl groups (38). Phosphorylation of lamin B by PKC, and p34 cdc2/cyclin B kinase, leads to nuclear lamina disassembly, a process required for entry into mitosis (38). Current studies are focused on determining the identity and mechanism of the cell cycle regulation of nuclear PI-PLC and on characterizing the mechanisms by which PG and the V5 region of PKC ␤ II participate, along with the C1 and C2 domains, in regulating nuclear PKC ␤ II activity.