Regulation of Protein Kinase Cν by the B-cell Antigen Receptor

Diacylglycerol-dependent signaling plays an important role in signal transduction through T- and B-lymphocyte antigen receptors. Recently, a novel serine-threonine kinase of the protein kinase C (PKC) family has been described and designated as PKCν. PKCν has two putative diacylglycerol binding C1 domains, suggesting that it may participate in a novel diacylglycerol-mediated signaling pathway. Here we show that both endogenous and recombinant PKCν are trans-located to the plasma membrane and activated by the diacylglycerol mimic phorbol 12-myristate 13-acetate. Mutational analysis demonstrates that PKCν activation is dependent on trans-phosphorylation of two conserved activation loop serine residues. We also find that PKCν is an important physiologic target of the B-cell receptor (BCR), because PKCν is found to be abundantly expressed in chicken and human B-cell lines and, in addition, exhibits robust activation after BCR engagement. Genetic and pharmacologic analyses of BCR-mediated PKCν activation indicate that it requires intact phospholipase Cγ and PKC signaling pathways. Furthermore, in co-transfection assays, PKCν can be trans-phosphorylated by the novel PKC isozymes PKCε, PKCη, or PKCθ but not the classical PKC enzyme, PKCα. Taken together, these results suggest that PKCν is an important component of signaling pathways downstream from novel PKC enzymes after B-cell receptor engagement.

One of the earliest detectable events following engagement of lymphocyte antigen and Fc receptors is activation of the phospholipase C isozyme ␥ (PLC␥) 1 (reviewed in Refs. [1][2][3][4][5]. Activated PLC␥ acts to hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate, resulting in the generation of the second messengers diacylglycerol (DAG) and inositol 3,4,5trisphosphate. Soluble inositol 3,4,5-trisphosphate diffuses through the cytoplasm to bind to and gate inositol 3,4,5trisphosphate receptor ion channels expressed on intracellular calcium store membranes, thereby initiating a general increase in cytosolic Ca 2ϩ , which is a critical component of antigen and Fc receptor cell activation signals (reviewed in Refs. 6 -9 and by others). In contrast, DAG remains associated with cellular membranes and serves as an essential cofactor in the assembly of a functional "signalsome" in the subplasmalemmal region beneath engaged receptors. Whereas a large body of evidence from studies of PLC␥ and PKC signaling indicates that the DAG-dependent component of antigen and Fc receptor signals influence diverse aspects of immune cell biology (2, 10 -16), understanding the molecular mechanisms through which DAG acts requires a detailed knowledge of the direct targets of DAG and how they are influenced by the production of DAG following receptor engagement.
A novel serine-threonine kinase with two potential DAGbinding C1 domains has recently been cloned and designated PKC, but its activation mechanism and the identity of cell surface receptors that utilize its signaling capacity remain uncharacterized. As our initial analyses indicated that PKC is abundantly expressed in human B-cells, we investigated whether PKC was involved in signals mediated by the B-cell antigen receptor (BCR). Utilizing a combination of biochemical, genetic, and pharmacologic approaches, here we show that PKC is a downstream effector for BCR-mediated DAG production and that its activation mechanism probably involves DAGmediated membrane trans-location followed by trans-phosphorylation of two conserved residues within its "activation loop" by novel PKC enzymes.

MATERIALS AND METHODS
Reagents-Constitutively active PKC mutants were obtained from David Rawlings (Department of Pediatrics, University of Washington) and Peter Parker (Protein Phosphorylation Laboratory, Cancer Research UK). An M2 FLAG monoclonal antibody covalently coupled to agarose beads was from Sigma. A polyclonal antibody recognizing the carboxyl-terminal 16 residues of human and chicken PKC (HFIMAP-NPDDMEEDP) was generated by standard immunological techniques and affinity-purified against the immunizing peptide. Monoclonal PKC antibodies and a V5 epitope antibody were from Transduction Laboratories and Invitrogen, respectively. A polyclonal antibody that specifically recognizes a phosphorylated serine 735 residue in the activation loop of PKC was obtained from Doreen Cantrell (Lymphocyte Activation Laboratory, Cancer Research UK). This antibody is directed against a phosphorylated epitope that is conserved in all three members of the PKD kinase family. F(abЈ) 2 fragments of anti-human IgM were from Jackson Laboratories. A monoclonal-stimulating antibody recognizing the chicken BCR (M4) was purified from hybridoma supernatant using standard procedures. The classical/novel PKC inhibitor Ro-31-8025 was from Calbiochem. All of the other reagents were from standard suppliers or as indicated in the text.
Transient transfection of HEK 293 cells was carried out using a Beckman Gene-Pulser electroporation apparatus. 1 ϫ 10 7 cells/0.5 ml serum-free media were pulsed in 0.4-cm cuvettes with 10 g of plasmid DNA at 330 volts and 1000 microfarads before diluting with 10 ml of complete medium. Cells were allowed to recover overnight before experimental use. For transfection of A20 B-cells, the electroporation conditions used were 250 volts and 950 microfarads. cDNA Cloning and Mutagenesis-The PKC coding sequence was PCR-amplified from a human brain cDNA library (Clontech) using 5Ј-ACGTGCGGCCGCTGTCTGCAAATAATTCCCCTCCATCAGCCCA-G-3Ј forward and 5Ј-ACGTTCTAGATTAAGGATCTTCTTCCATATCA-TCTGGATTAGG-3Ј reverse primers. The PCR fragment was subcloned NotI/XbaI (sites are underlined) into a modified pcDNA4/TO doxycycline-inducible mammalian expression vector. This modified vector contains an in-frame FLAG epitope coding sequence, resulting in the expression of an amino-terminally tagged FLAG-PKC protein. A similar method was used to construct the pcDNA4/TO FLAG-PKD vector. To generate the pcDNA4/TO GFP-PKC construct, the coding sequence for GFP was PCR-amplified with 5Ј-HindIII and 3Ј-NotI restriction sites. This PCR fragment was then cloned into the pcDNA4/TO vector, and the PKC coding sequence was cloned in-frame COOH-terminal to the GFP sequence using NotI and XbaI restriction sites. PKC␣, PKC⑀, and PKC mutants were cloned into a modified pcDNA5/TO vector with an in-frame amino-terminal V5 epitope tag.
Site-specific mutations within the catalytic domain of PKC, resulting in single or double amino acid substitutions, were generated by overlap PCR using wild-type PKC as the template. Mutants were generated using the above primers together with internal forward and reverse primers complementary to each other and containing specific nucleotide substitutions as required. Primers (forward sequence only shown) containing the desired mutation(s) (underlined) were as follows: PKC-K605N, 5Ј-GGGAGGGATGTGGCTATTAACGTAATTGATAAG-ATGAG-3Ј; PKC-S731A/S735A, 5Ј-CATTGGTGAAAAGGCATTCAGG-AGAGCTGTGGTAGGAACTCCAGC-3Ј; and PKC-S731E/S735E, 5Ј-CATTGGTGAAAAGGAATTCAGGAGAGAGGTGGTAGGAACTCCA-GC-3Ј.
Following the second PCR reaction, the amplified cDNAs were subcloned (NotI/XbaI) into the modified pcDNA4/TO expression vector. Constructs were sequenced using an Applied Biosystems automated DNA sequencer before they were used in transient expression experiments. Protein expression was induced by treating cells with 5 g/ml doxycycline for 24 h.
Cell Fractionation-DT40 B-cells were washed in ice-cold phosphatebuffered saline and resuspended in 1 ml of ice-cold fractionation buffer (10 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, protease inhibitors, 1 mM AEBSF). Cells were lysed by homogenization, and unbroken cells/nuclear debris were removed by centrifugation at 800 ϫ g for 10 min at 4°C. The supernatant was subjected to high speed ultracentrifugation at 100,000 ϫ g for 30 min at 4°C, resulting in a soluble cytosolic fraction and an insoluble membrane pellet. The membrane pellet was solubilized in 1 ml of fractionation buffer containing 1% Triton-X for 20 min at 4°C before insoluble material was removed by centrifugation at 20,000 ϫ g for 10 min at 4°C. PKC was then immunoprecipitated from both cytosolic and membrane fractions and analyzed by Western blotting.
In Vitro Kinase Assays-Immunocomplexes were washed twice in lysis buffer (described above) and once in kinase buffer (30 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 ). PKC autophosphorylation was determined by incubating immunocomplexes with 20 l of kinase buffer containing 100 M [␥-32 P]ATP at 30°C for 10 min. Reactions were terminated by the addition of 2ϫ SDS-PAGE sample buffer, and the samples were analyzed by 8% SDS-PAGE and autoradiography.
Microscopy-For immunofluorescent localization of endogenous PKC, DT40 B-cells were resuspended in phosphate-buffered saline and allowed to attach to polylysine-coated glass bottom dishes (MatTek Inc.). The cells were then left untreated or treated with 50 ng/ml PMA for 10 min before fixing with 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with a 0.5% saponin buffer and sequential incubation with primary (anti-PKC, 1 g/ml) and secondary antibodies (anti-rabbit Alexa Fluor 488, 1:3000 dilution) for 20 min. After each step, the cells were washed three times in phosphate-buffered saline containing 1% bovine serum albumin. For GFP visualization, A20 B-cells transiently expressing GFP-PKC were plated on polylysine-coated glass bottom dishes in phosphate-buffered saline and allowed to adhere before stimulation with 50 ng/ml PMA. The cells were excited with a 495-nm wavelength light, and emitted light was imaged using an IMAGO CCD camera set for a 2-s exposure using a Zeiss microscope and TillVision software. All of the experiments presented a representative of two to three independent experiments.

RESULTS
Although the PKC gene and transcripts have been described previously (17), there is no present literature regarding its regulation or receptor systems that utilize PKC as a signaling mechanism. However, PKC has two putative C1 domains. These domains (see schematic in Fig. 1A) of ϳ50 residues are thought to bind the lipid second messenger diacylglycerol, suggesting that PKC might participate in a diacylglycerol-mediated signaling pathway. As the tumor-promoting phorbol esters serve as pharmacological substitutes for DAG and mimic many aspects of the biological activity of DAG, we used one of them, PMA, to evaluate the potential involvement of PKC in DAG signaling by imaging the subcellular localization of PKC in cells that had been left untreated or treated with PMA (Fig. 1B). As can be seen, both endogenous PKC (imaged in fixed and antibody-stained cells, top panels) and GFP-tagged PKC (imaged in live cells, bottom panels) are substantially redistributed from the cytosol to the plasma membrane in response to PMA treatment, consistent with DAG serving as a membrane recruitment signal for PKC.
Recruitment to the plasma membrane often serves as a means for activation of protein kinases, and PMA has previously been shown to induce both membrane recruitment and activation of PKD1, one of the closest homologues of PKC (18,19). To understand how membrane recruitment affects PKC function, we produced a FLAG-tagged PKC construct as a backbone for mutational analysis of PKC activation and confirmed its expression after transfection of HEK 293 cells (Fig.  1C, left panel). The treatment of cells expressing FLAG-PKC with PMA induced easily detectable enzymatic activation as measured by an in vitro kinase assay (Fig. 1C, right panel, top  blot). For many serine-threonine kinases, phosphorylation within the activation loop serves as a marker for enzymatic activation. The putative activation loop residues of PKC are serines 731 and 735. Therefore, we utilized an antibody targeted specifically at phosphoserine 735 and its surrounding region to analyze activation loop phosphorylation of FLAG-PKC. Anti-pS735 immunoreactivity strongly correlated with the activation state of FLAG-PKC, suggesting a role for phosphorylation of this residue during PKC activation (Fig. 1C, The cells were left untreated or treated with PMA, and expressed proteins were analyzed by anti-FLAG immunoprecipitation followed by either in vitro kinases assays (measuring PKC autophosphorylation) or by anti-pS735 and anti-FLAG immunoblotting. Note that the anti-pS735 immunoreactivity together with the lack of kinase activity of the PKC-KN mutant demonstrates that a significant fraction of anti-pS735 immunoreactivity of stimulated PKC is because of a transphosphorylation event. B, phosphorylation of activation loop serines is necessary and sufficient for PKC activation. HEK 293 cells were transiently transfected with pcDNA4/TO vectors driving the expression of the indicated constructs, treated with doxycycline to induce protein expression, and treated or not treated with PMA. Cells were lysed, and expressed proteins were analyzed by anti-FLAG immunoprecipitation followed by in vitro kinase assay and anti-FLAG immunoblotting. SS/ EE, S731E/S735E; SS/AA, S731A/S735A.

FIG. 3. B-cell receptor engagement induces activation of endogenous PKC in chicken and human B-cell lines.
A, chicken DT40 B-cells were treated or not treated with anti-chicken IgM, lysed at the indicated times, immunoprecipitated with anti-PKC, and analyzed by anti-pS735 and PKC immunoblotting. B, human Raji and Ramos B-cell lines were treated or not treated with F(abЈ) 2 fragments of antihuman IgG, lysed at the indicated times, immunoprecipitated with anti-PKC, and analyzed by anti-pS735 and PKC immunoblotting. C, chicken DT40 B-cells were left untreated (Ϫ) or were treated with either anti-chicken IgM (BCR) or with 50 ng/ml PMA for 3 min as indicated. Cytosolic and membrane fractions were prepared as described under "Experimental Procedures," and PKC activity was analyzed by anti-pS735 and PKC immunoblotting. right panel, middle blot). We further utilized anti-pS735 to examine at the activation of endogenous PKC. As our initial analyses (data not shown) had indicated the presence of abundant PKC in several B-cell lines, we evaluated whether PMA treatment could activate endogenous PKC in chicken DT40 B-cells and the Raji and Ramos human B-cell lines (Fig. 1D). As can be seen, PMA treatment strongly induced anti-pS735 immunoreactivity of anti-PKC immunoprecipitates, indicating that endogenous PKC is activated by PMA in the same manner as the recombinant FLAG-PKC.
To further investigate the role of pS735 phosphorylation in PKC activation, we constructed a kinase-deficient mutant of PKC on the FLAG-PKC backbone via the mutation of a conserved lysine residue within the putative ATP-binding cassette of the kinase domain (FLAG-PKC-KN). This mutant had no detectable kinase activity as assessed by an in vitro kinase assay ( Fig. 2A, top panel). However, a comparison of the anti-pS735 immunoreactivity induced by PMA treatment of wildtype FLAG-PKC with that of the FLAG-PKC-KN mutant demonstrated essentially intact phosphorylation of this site ( Fig. 2A), indicating that this site is trans-phosphorylated by an upstream kinase in intact cells. In some proteins whose function is modulated by phosphorylation at serine or threonine residues, the replacement of the regulatory serine or threonine residues with negatively charged glutamate or aspartate residues induces the protein to act as if it is constitutively phosphorylated at the mutated sites. Conversely, the replacement with alanine produces a protein whose function can no longer be modulated by phosphorylation. Therefore, we further analyzed the activation mechanism PKC by producing mutants on the FLAG-PKC backbone with potentially activating mutations at positions 731 and 735 (serine to glutamate, S731E/S735E) or deactivating mutations (serine to alanine at the same positions, S731A/S735A) and analyzing their responses to PMA treatment (Fig. 2B). Although the S731A/ S735A mutant is no longer activated by PMA, the S731E/ S735E mutant shows high constitutive activity that is PMAindependent, indicating that phosphorylation at serines 731 and 735 is both necessary and sufficient for PKC activation. When viewed in conjunction with the redistribution to the plasma membrane induced by PMA treatment, a compelling model for PKC activation can be constructed in which its activation occurs as the result of its membrane trans-location and subsequent trans-phosphorylation by an upstream PMAregulated protein kinase on serine 731/735.  Fig. 3C, PKC translocates from the cytosol to the membrane fraction in response to PMA treatment (Fig. 3C, lower panels), consistent with the observation that PMA induces the trans-location of GFP-PKC from the cytosol to the plasma membrane (see Fig. 1B). In addition, pS735 immunoblotting reveals that activated PKC is restricted to the membrane fraction of PMA-treated B-cells (Fig. 3C, upper panels). In contrast, a portion of PKC translocates to the membrane fraction of BCR-stimulated B-cells, and activated PKC is detectable in both the cytosolic and membrane fractions (Fig. 3C). Kinetic analysis indicates that PKC rapidly redistributes from the cytosol to the membrane compartment of B-cells in response to BCR ligation (within Ͻ30 s) and that activated PKC is found in cytosolic and membrane compartments both at early (Ͻ30 s) and late (Ն10 min) time points (data not shown).
That the activation of PKC by BCR ligation is entirely dependent on PLC␥ activation was demonstrated through the use of DT40 B-cell lines engineered to be deficient in individual components of the signaling cascade, leading to PLC␥ activation (Fig. 4A). Lyn-deficient DT40 cells have intact but delayed PLC␥ activation (20). They also exhibit relatively intact but delayed activation of PKC (peak activation occurs at Ͼ10 min as opposed to ϳ1 min in wild-type DT40 cells). This closely tracks the published time course of PLC␥ activation as measured by inositol phosphate turnover (20). In contrast, DT40 cell lines deficient in BLNK, BTK, and PLC␥ 2 , each of which have completely abrogated PLC␥ activation (reviewed in Ref. 21), show completely abrogated PKC activation. Note that in addition PMA-mediated PKC activation is intact in all of the DT40 cell lines tested, eliminating the possibility that direct effects of the deficiency of these proteins might be affecting PKC activation. Consistent with these results (Fig. 4B), the treatment of cells with the putative DAG antagonist calphostin C also abrogated BCR-mediated PKC activation in chicken and human B-cells.
The above results demonstrate that PLC␥ is a probable source of DAG for BCR-mediated PKC activation. Because DAG would plausibly serve to membrane target and activate both classical and novel PKC enzymes in the same general microdomain area(s) as PKC would be localized, we investigated whether either of these classes of enzymes might serve as an upstream activating kinase for PKC. Consistent with this possibility, the treatment of B-cells with the classical/novel PKC inhibitor Ro-31-8025 completely blocked BCR-mediated PKC activation (Fig. 5A). Whereas this inhibitor is thought to be relatively specific for the classical and novel classes of PKC enzymes relative to other serine/threonine kinases (including PKD1, the closest homologue of PKC (22)), the use of inhibitors is always open to questions regarding specificity within the cellular environment. Therefore, to further evaluate the role of PKC-dependent trans-phosphorylation as an activation mechanism for PKC, we tested the ability of activated mutants of various PKC subtypes to induce PKC phosphorylation (and thus activation) in a heterologous expression assay. The expression of constitutively activated mutants of novel PKC isozymes (, ⑀, and ) produced robust constitutive activation of PKC in the absence of PMA stimulation (Fig. 5B). In contrast, the co-expression of kinase-deficient or wild-type PKC⑀ or PKC had little or no effect on basal or PMA-induced activation of PKC. Interestingly, the expression of a constitutively activated classical PKC enzyme, PKC␣, produced no detectable change in either constitutive or PMA-induced PKC activation (Fig. 5C), suggesting that PKC is a poor substrate for PKC␣ and potentially other classical PKC isoforms. DISCUSSION We have analyzed the activation mechanism of the novel serine-threonine kinase PKC and show that PKC is activated by PMA and BCR-mediated DAG production via the transphosphorylation of two serine residues (Ser 731 and Ser 735) within its activation loop. The ability of activated mutants of novel PKC isozymes but not the classical PKC enzyme, PKC␣, to induce constitutive PKC activation suggests that this transphosphorylation event may be mediated primarily by novel PKC enzymes. As PKC exhibits robust activation in response to BCR engagement, our results suggest that PKC is an important downstream target of activated novel PKC enzymes during BCR signaling.
The closest homologues of PKC are PKD1/PKC and PKD2, and together these three kinases form a distinct protein kinase subfamily. They share a predicted tertiary structure that includes two C1 domains contained in their amino-terminal halves, a single central pH domain, and closely homologous kinase domains in their COOH-terminal halves. Consistent with their structural similarity to PKC, PKD1 and PKD2 (similar to PKC) appear to act downstream from both DAG and protein kinase C enzymes. Although the data in this paper suggest that PKC appears to relatively specifically targeted by novel PKC isoforms, PKD1 and PKD2 are activated by both classical and novel PKCs (19,(23)(24)(25). From the standpoint of their catalytic domains, these three kinases are only distantly related to the ACG kinases (consisting of the PKA, PKC, and PKG protein kinase families). Instead, their kinase domains exhibit the closest sequence similarity to those of calciumregulated kinases (25). Consistent with this finding, small peptides phosphorylated by PKD1 in vitro do not appear to significantly overlap with those phosphorylated by classical/novel PKC enzymes (23,26), suggesting that PKC/PKD1/PKD2 substrates represent distinct signaling pathways downstream from DAG and PKCs.
Whether PKD1, PKD2, and PKC share similar substrate ranges or downstream biological effector functions remains to be demonstrated. However, their position downstream from novel PKCs suggests that one or more of them is involved in linking novel PKC activation with effector responses downstream from the BCR in B-cells. In this regard, a recent report (14) has implicated the novel PKC isoform PKC␦ in controlling the mechanisms of anergy and tolerance in B-cells. Because PKD1 and PKC are both expressed in B-cells and appear to be targets of novel PKC enzymes, either one or both could plausi- FIG. 5. Novel PKC isoforms control the phosphorylation and thus activity of PKC in intact cells. A, the classical/novel PKCspecific inhibitor Ro-31-8425 blocks PKC activation. Wild-type DT40 B-cells were left untreated or were treated with 5 M of the classical/ novel PKC inhibitor Ro-31-8425 prior to PMA (P) or BCR (for the times indicated) stimulation. Cells were lysed, and endogenous PKC was immunoprecipitated (IP) and analyzed by Western blotting with the indicated antibodies. DMSO, Me 2 SO. B, top and middle panels, HEK 293 cells were co-transfected with pcDNA4/TO FLAG-PKC and either a control vector or different novel PKC expression constructs as indicated. KD, kinase-deficient; DA, dominant active; wt, wild-type. Cells were treated or not treated with PMA and lysed, and endogenous PKC was immunoprecipitated and analyzed by Western blotting with the indicated antibodies. Bottom panels, the expression of the various novel PKC enzymes was confirmed by Western blotting with anti-PKC antibodies or antibody against a V5 epitope tag. C, top and middle panels, HEK 293 cells were co-transfected with pcDNA4/TO FLAG-PKC and a control vector, a dominant active PKC␣ construct, or a dominant active PKC expression construct as indicated. Cells were treated or not treated with PMA and lysed, and endogenous PKC was immunoprecipitated and analyzed by Western blotting with the indicated antibodies. Bottom panel, the expression of the PKC␣ DA mutant protein was confirmed by Western blotting with an anti-PKC␣ antibody. bly function as a link between PKC␦ (and possibly other novel PKC enzymes) and downstream effectors and mechanisms involved in the creation of B-cell energy and tolerance. Determining whether PKC and/or its homologues operate in this pathway or an alternative signaling pathway will depend on the future development of genetic or pharmacologic tools for the manipulation of their signaling function.