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

Yesim Gökmen-PolarDagger and Alan P. FieldsDagger §parallel

From the Dagger  Sealy Center for Oncology and Hematology and the Departments of § Pharmacology and  Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1048

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In human erythroleukemia (K562) cells, the highly related protein kinase C (PKC) alpha  and PKC beta II isozymes serve distinct functions in cellular differentiation and proliferation, respectively. Previous studies using two domain switch PKC chimera revealed that the catalytic domains of PKC alpha  and beta II 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 beta II function. A cellular assay for PKC beta II function was devised based on the finding that PKC beta II 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 beta II or a PKC chimera that functions like PKC beta II. We demonstrate that a PKC alpha /beta II chimera containing only the carboxyl-terminal 13 amino acids from PKC beta II (beta II V5) is capable of nuclear translocation and lamin B phosphorylation. These results are consistent with our recent observation that the PKC beta II V5 region binds to phosphatidylglycerol (PG), a potent and selective PKC beta II activator present in the nuclear membrane (Murray, N. R., and Fields, A. P. (1998) J. Biol. Chem. 273, 11514-11520). Soluble beta II V5 peptide selectively inhibits PG-stimulated PKC beta II activity in a dose-dependent fashion, indicating that PG-mediated activation of PKC beta II involves interactions with the beta II V5 region of the enzyme. We conclude that beta II V5 is a major determinant for PKC beta II nuclear function and suggest a model in which binding of PG to the beta II V5 region stimulates nuclear PKC beta II activity during G2 phase of the cell cycle.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (1-4). 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 alpha  and beta 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 alpha  is involved in PMA-induced cytostasis and megakaryocytic differentiation (10). PKC beta II, on the other hand, is required for K562 cell proliferation (10). PKC beta II selectively translocates to the nucleus during the G2/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, 11-15).

In order to study the molecular basis of PKC alpha  and beta II isozyme function, we expressed two domain switch chimeras between the regulatory and catalytic domains of PKC alpha  and beta II in K562 cells (16). These chimeras demonstrated that the catalytic domains of PKC alpha  and beta II contain determinants that are critical for isozyme-specific function (16). Specifically, a PKC beta II/alpha chimera, consisting of the regulatory domain of PKC beta II and the catalytic domain of PKC alpha , exhibited a phenotype resembling wild type PKC alpha  (16). Conversely, a PKC alpha /beta II chimera, composed of the regulatory domain of PKC alpha  and the catalytic domain of PKC beta II, behaved like wild type PKC beta II (16).

Based upon these findings, we wished to define the isozyme-specific determinants within the catalytic domain of PKC beta II. For this purpose, we constructed and expressed a series of alpha /beta II PKC chimeras in which the variable and constant regions within the catalytic domain of PKC beta II were replaced by the corresponding PKC alpha  regions. Our results demonstrate that the V5 region of PKC beta II (beta II V5), consisting of the carboxyl-terminal 13 amino acids of PKC beta 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 beta II binds phosphatidylglycerol (PG), a nuclear activator of the enzyme (17). A soluble peptide corresponding to beta II V5 inhibits PG-stimulated PKC beta II activity, indicating that interactions between PG and the beta II V5 region are important for PG-mediated activation of PKC beta II. Based on these observations, we propose a model for the cell cycle-regulated activation of nuclear PKC beta II.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of PKC Chimeras-- In previous work, we demonstrated that a chimera consisting of the regulatory domain of PKC alpha  (alpha V1-V3) and the catalytic domain of PKC beta II (beta II C3-V5) behaved like PKC beta II (16). Therefore, we constructed PKC alpha /beta II chimeras in which each of the constant and variable regions within the catalytic domain of this chimera was replaced with the corresponding PKC alpha  sequence (Fig. 1). 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 alpha  and PKC beta  portions of the chimera using the appropriate 5' and 3' end primers, internal chimeric primers, and either PKC alpha  or PKC beta 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 5' and 3' 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 (5' KpnI and 3' 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).

                              
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Table I
PCR primers used to construct PKC chimeras

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 alpha  and beta 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 × 105 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 alpha  and beta 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% Me2SO). Previous studies demonstrated that this PMA treatment leads to loss of immunologically detectable PKC beta 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 alpha  V5 and beta 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 beta II Activity in Vitro-- In vitro protein kinase C assays were carried out using purified baculovirus-expressed human PKC beta II as described previously (15). Briefly, reactions were performed in assay buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgSO4, 1 mM dithiothreitol, 100 µM CaCl2, 100 µM ATP, 2 µCi of [gamma -32P]ATP (Amersham Pharmacia Biotech), 40 µg/ml (50 µM) phosphatidylserine (PS, Avanti Polar Lipids), 20 µM dioctanoylglycerol (DAG, Avanti Polar Lipids), and 10 µg of histone H1 for 15 min at 25 °C. In most reactions, 250 µg/ml (315 µM) dioleoyl PG (Avanti Polar Lipids) was added to maximally stimulate PKC beta II activity (17). Synthetic peptides corresponding to beta II V5 (CFVNSEFLKPEVKS) or alpha  V5 (CQFVHPILQSSV) were added at the concentrations indicated in the figure legends. Histone H1 phosphorylation was quantitated using phosphorimaging analysis as described previously (12).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction and Expression of PKC Chimeras in K562 Cells-- Based on our previous finding that the catalytic domain of PKC beta II is crucial for PKC beta II function (16), we generated a series of PKC chimeras in which the variable and constant regions within the catalytic domain of PKC beta II were exchanged with the corresponding sequences from PKC alpha  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 alpha  and beta II (Fig. 2). Expression of each of the chimeras containing the beta II V5 region (beta II V4, beta II C4, beta II V5, beta II 1504, and beta II 1851) was confirmed using the PKC beta II V5 antibody (Fig. 2A). Expression of the alpha  beta II alpha  chimera, which contains the carboxyl-terminal V5 region from PKC alpha , was confirmed using the PKC alpha  V5 antibody (Fig. 2B). In each case, a band with apparent molecular mass of 85 kDa was detected, corresponding in size to intact PKC alpha  and beta 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 alpha  or PKC beta II. The chimeric nature of each of the chimeras was confirmed by immunoblot analysis using the appropriate PKC alpha  and beta II-specific V3 antibodies as described previously (Ref. 16; data not shown).


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Fig. 1.   Schematic representation of the constructed PKC alpha /beta II chimeras. PKC chimeras were constructed between PKC alpha  and beta II by a two-step PCR method as described under "Experimental Procedures." The schematic indicates the regulatory and catalytic domains of PKC. The conserved (C1-C4) and variable regions (V1-V5) regions of PKC are also indicated. Open boxes represent PKC alpha  sequences, and hatched boxes correspond to PKC beta II sequences.


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Fig. 2.   Expression of PKC chimeras in K562 cells. Total cell lysates from 1 × 105 K562 cells, transfected with vector containing the indicated PKC chimera, were subjected to immunoblot analysis as described under "Experimental Procedures." Panel A, immunoblot detection using isotype-specific antibody against the V5 region of PKC beta II. Cell transfectants expressing the following expression vector constructs were subjected to immunoblot analysis: pREP4 control vector (- Control), wild type PKC beta II (+ Control), beta II V4, beta II C4, beta II V5, alpha  beta II alpha , beta II 1504, and beta II 1851 PKC chimeras. Panel B, immunoblot detection using isotype-specific antibody against the V5 region PKC alpha . Lane -Control, pREP4 vector; lane +Control, wild type PKC alpha ; alpha beta IIalpha , PKC chimera.

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 alpha  expression and a loss of PKC beta 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 beta II expression was dramatically reduced as a consequence of PMA treatment (Fig. 3A, compare lanes 1 and 2) (10, 16). In contrast, the levels of PKC alpha  were increased as previously reported (10, 16) (Fig. 3B, compare lanes 1 and 2). Interestingly, in contrast to endogenous PKC beta II, the expression of each of the transfected PKC chimeras (beta II V4, beta II C4, beta II 1504, beta II 1851, beta II V5, and alpha beta IIalpha ) persists after PMA treatment (Fig. 3, A and B).


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Fig. 3.   Expression of PKC chimeras persists after PMA-treatment in K562 cells. Panel A, K562 cells transfected with either control vector or PKC chimera vectors were treated with 30 nM PMA for 48 h. Following 48 h treatment, total cell lysates from 1 × 105 cells were immunoblotted with antibody against the V5 region of PKC beta II as described under "Experimental Procedures." Lane Control (- PMA), pREP4 vector in the absence of PMA; lane Control (+ PMA), pREP4 in the presence of PMA; lanes beta IIV4, beta IIC4, alpha beta IIalpha ; beta II1504, beta II1851, and beta IIV5, PKC chimeras after PMA treatment. Panel B, immunoblot analysis as described under "Experimental Procedures" with antibody against the V5 region of PKC alpha . Lanes: controls are the same as in A; alpha beta IIalpha , PKC chimera after PMA treatment.

Ability of PKC Chimeras to Reconstitute PKC beta II Function-- The observation that expression of PKC chimera persists after chronic PMA treatment while endogenous PKC beta II levels are dramatically reduced suggested a strategy to specifically determine whether the transfected PKC chimeras could function like PKC beta II in intact K562 cells without the contribution of endogenous PKC beta II. This strategy is predicated upon several key observations. First, PKC beta 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 beta II to the nucleus leads to direct phosphorylation of the nuclear envelope polypeptide lamin B (11-15). Third, the ability of bryostatin to stimulate nuclear lamin B phosphorylation is strictly dependent upon the expression of PKC beta II (16). Thus, when K562 cells are treated with PMA, both PKC beta 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 (-PMA) and Control (+PMA). K562 cells expressing the beta II V4, beta II C4, beta II 1504, beta II 1851, and beta II V5 chimeras were all capable of reconstituting bryostatin-mediated lamin B phosphorylation after treatment with PMA. In contrast, cells expressing the alpha beta IIalpha chimera were incapable of reconstituting the bryostatin-mediated response. From these results, we conclude that chimeras containing the functional determinant important for PKC beta II function are capable of reconstituting PKC beta II-dependent lamin B phosphorylation. Furthermore, our analysis localizes this functional determinant to the extreme carboxyl-terminal 13 amino acids of PKC beta II (beta II V5), since a chimera containing only these 13 amino acid residues from PKC beta II (PKC beta II V5) is capable of functioning in this PKC beta II-selective pathway.


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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.

PG-stimulated PKC beta II Activity Involves Interactions with the V5 Region of PKC beta II-- The results from the PKC chimera studies demonstrate that the carboxyl-terminal V5 region of PKC beta II represents a molecular determinant for PKC beta 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 activates PKC beta II (19). More recently, we identified NMAF as PG (17). PG was found to be a potent and selective activator of PKC beta 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 beta II (17), suggesting that PG stimulates PKC beta II activity through interactions involving beta II V5. To specifically test this hypothesis, we assessed the effect of soluble beta II V5 peptide on PG-stimulated PKC beta II activity in vitro (Fig. 5). As reported previously (17) addition of 315 µM PG stimulates PKC beta 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 µM PS (data not shown). Inclusion of beta II V5 peptide in the kinase assay leads to inhibition of PG-stimulated PKC beta II histone kinase activity (Fig. 5A, lane 5). Inhibition is specific for the beta II V5 peptide, since no inhibition is observed using the corresponding V5 peptide from PKC alpha  (Fig. 5A, lane 4). Inhibition by beta II V5 peptide is dose-dependent with an apparent IC50 of ~100 µM (Fig. 5B), whereas significant inhibition was not observed with the corresponding alpha  V5 peptide. Interestingly, the beta II V5 peptide inhibits only the PG-stimulated component of PKC beta 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 beta II, whereas calcium, DAG, and PS mediate activation by binding to the C1 and C2 regions within the regulatory domain of the enzyme.


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Fig. 5.   PG-stimulated PKC beta II activity is inhibited by the beta II V5 peptide in a dose-dependent manner. Recombinant human PKC beta II was incubated in kinase buffer in the absence or presence of the indicated concentration of either beta II V5 or alpha V5 peptide for 15 min at 25 °C as described under "Experimental Procedures." Activity was monitored by incorporation of 32P into purified histone H1 (10 µg) as described previously (12). Panel A, autoradiograph of PKC beta II-mediated histone H1 phosphorylation by PKC beta II. Reactions were as follows. Lane 1, no PKC beta II added; lane 2, PKC beta II in reaction buffer without PG; lane 3, PKC beta II in reaction buffer with PG; lane 4, PKC beta II in reaction buffer containing PG and 1 mM alpha V5 peptide; lane 5, PKC beta II in reaction buffer containing PG and 1 mM beta II V5 peptide. Data are from a representative experiment. Panel B, dose-dependent inhibition of PG-stimulated PKC beta II activity with either beta II V5 (open circles) or alpha V5 (closed circles) peptide. Results are plotted as the percentage of the PG-stimulated PKC beta II activity versus peptide concentration (µM). Data represent the mean of five determinations ± S.D. Some error bars are masked by the data point symbols.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha , beta II, and iota , and each of these isozymes serve distinct functions in these cells. PKC alpha  is important in cellular differentiation (10), PKC beta II is required for cellular proliferation (10) and PKC iota  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 beta II. PKC beta II is required for cell cycle progression through the G2/M phase transition (12, 13). PKC beta II is activated at the nucleus during late G2 phase just prior to mitosis in synchronized cells (12, 13). At the nucleus, PKC beta 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 beta II activation leads to cell cycle arrest in G2 phase, demonstrating the importance of nuclear PKC beta II activation and lamin B phosphorylation in entry into mitosis (13).

Nuclear PKC beta 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 G2 phase that serves to activate nuclear PKC beta II (21). Second, the nuclear membrane contains a potent activator of PKC beta 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 beta II activation suggested that PG might function through interactions within the carboxyl-terminal V5 region of PKC beta II. Indeed, we have demonstrated that the carboxyl-terminal region of PKC beta 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 beta II that allow it to translocate to the nucleus and phosphorylate lamin B in intact cells, a process that we have demonstrated requires PKC beta II expression (16). We previously demonstrated that a chimera between PKC alpha  and PKC beta II containing the catalytic domain of PKC beta II was capable of translocating to the nucleus and phosphorylating lamin B (16). In contrast, a chimera containing the catalytic domain of PKC alpha  was not capable of mediating this response (16). From these data, we concluded that regions within the catalytic domain of PKC beta II serve as a molecular determinant of PKC beta II function (16). We have now used further PKC alpha /beta II chimeras to map this molecular determinant to the extreme carboxyl-terminal 13 amino acids of PKC beta II. The results of this study are interesting in light of our recent finding that the V5 region of PKC beta II mediates binding to PG (17). In fact, we now demonstrate that a beta II V5 peptide selectively inhibits PG-stimulated PKC beta II activity in a dose-dependent fashion. These results indicate that interactions between PG and beta II V5 are important for PG- stimulated PKC beta II activity.

The identification of the carboxyl-terminal region of PKC beta 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 alpha  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 alpha  to appropriate intracellular sites where it can transduce isozyme-specific signals (22). Likewise, we have demonstrated that interaction of the V5 region of PKC beta 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 beta I and beta II activity by calcium have led to the suggestion that the V5 region of PKC beta 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 beta II, the proposed interdomain interactions involving the C2 and V5 regions of PKC beta 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 beta II as illustrated in Fig. 6. Soluble PKC beta II is stably phosphorylated on at least three known sites (24, 25). First, PKC beta 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 beta 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 G2 phase of cell cycle (21). Activation of nuclear PI-PLC leads to generation of inositol trisphosphate (IP3) and DAG. We propose that IP3 binds to nuclear IP3 receptors to mobilize intracellular calcium stores, leading to a rise in intracellular calcium concentrations. It has been demonstrated that nuclei contain functional IP3 receptors (26) and that intracellular calcium concentrations rise in synchronized cells just prior to mitosis (27-29). Furthermore, inhibition of this rise in intracellular calcium, using calcium chelators such as BAPTA, leads to cell cycle arrest in G2 phase, demonstrating the importance of calcium in entry into mitosis (29). We propose that one function of elevated calcium levels is to translocate PKC beta II to the inner nuclear membrane. We have not directly determined whether this step involves translocation of PKC beta II from cytosolic pools, or alternatively involves translocation of inactive PKC beta II from a nucleoplasmic pool. However, based on our fractionation studies (9, 11-15) and the immunofluorescence studies of others in cultured myocytes (30), we favor the hypothesis that this step represents translocation of cytosolic PKC beta 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 (31-33). 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 pseudosubstrate domain from the active site of the enzyme (37). It has recently been proposed that the V5 region of PKC beta 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 beta 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 beta 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 beta II participate, along with the C1 and C2 domains, in regulating nuclear PKC beta II activity.


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Fig. 6.   A proposed model for the nuclear activation of PKC beta II. Prior to activation, PKC beta 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 G2 phase of cell cycle, a nuclear PI-PLC is activated, leading to the generation of nuclear DAG (21) and IP3. IP3 activates nuclear IP3 receptors (26), leading to elevation of intranuclear calcium concentrations just prior to mitosis (27-29). 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 beta II, thereby inducing a conformational change that releases the pseudosubstrate domain of PKC beta II from the active site. The carboxyl-terminal region of the enzyme is brought into juxtaposition with the C2 domain via specific binding of beta II V5 to clustered PG, leading to full activation of the enzyme. Activated PKC beta II directly phosphorylates its nuclear substrate lamin B, leading to mitotic nuclear lamina disassembly and entry into mitosis (38).

    ACKNOWLEDGEMENTS

We thank B. Sun and Y. Wang for technical assistance in the construction of PKC chimeras, N. R. Murray for many helpful suggestions, and D. Schattenberg for preparation of the PKC model (Fig. 6).

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA56869.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel A Leukemia Society of America Scholar. To whom correspondence should be addressed: Sealy Center for Oncology and Hematology, University of Texas Medical Branch, Medical Research Bldg., Rm. 9.104, 301 University Blvd., Galveston, TX 77555-1048. Tel.: 409-747-1940; Fax: 409-747-1938; E-mail: afields{at}marlin.utmb.edu.

The abbreviations used are: PKC, protein kinase C; PG, phosphatidylglycerol; PS, phosphatidylserine; PCR, polymerase chain reaction; DAG, dioctanoylglycerol; IP3, inositol trisphosphatePMA, phorbol 12-myristate 13-acetateNMAF, nuclear membrane activation factorPI-PLC, phosphatidylinositol-specific phospholipase C.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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