Regulation of Protein Kinase C&ngr; by the B-cell Antigen Receptor*

Sharon A. MatthewsDagger , Rashmi DayaluDagger , Lucas J. ThompsonDagger , and Andrew M. ScharenbergDagger §

From the Dagger  Department of Pediatrics and Immunology, University of Washington and Children's Hospital and Regional Medical Center, Seattle, Washington 98195

Received for publication, November 5, 2002, and in revised form, December 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PKCnu . PKCnu 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 PKCnu are trans-located to the plasma membrane and activated by the diacylglycerol mimic phorbol 12-myristate 13-acetate. Mutational analysis demonstrates that PKCnu activation is dependent on trans-phosphorylation of two conserved activation loop serine residues. We also find that PKCnu is an important physiologic target of the B-cell receptor (BCR), because PKCnu 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 PKCnu activation indicate that it requires intact phospholipase Cgamma and PKC signaling pathways. Furthermore, in co-transfection assays, PKCnu can be trans-phosphorylated by the novel PKC isozymes PKCepsilon , PKCeta , or PKCtheta but not the classical PKC enzyme, PKCalpha . Taken together, these results suggest that PKCnu is an important component of signaling pathways downstream from novel PKC enzymes after B-cell receptor engagement.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

One of the earliest detectable events following engagement of lymphocyte antigen and Fc receptors is activation of the phospholipase C isozyme gamma  (PLCgamma )1 (reviewed in Refs. 1-5). Activated PLCgamma acts to hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate, resulting in the generation of the second messengers diacylglycerol (DAG) and inositol 3,4,5-trisphosphate. Soluble inositol 3,4,5-trisphosphate diffuses through the cytoplasm to bind to and gate inositol 3,4,5-trisphosphate receptor ion channels expressed on intracellular calcium store membranes, thereby initiating a general increase in cytosolic Ca2+, 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 PLCgamma 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 DAG-binding C1 domains has recently been cloned and designated PKCnu , but its activation mechanism and the identity of cell surface receptors that utilize its signaling capacity remain uncharacterized. As our initial analyses indicated that PKCnu is abundantly expressed in human B-cells, we investigated whether PKCnu 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 PKCnu is a downstream effector for BCR-mediated DAG production and that its activation mechanism probably involves DAG-mediated membrane trans-location followed by trans-phosphorylation of two conserved residues within its "activation loop" by novel PKC enzymes.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PKCnu (HFIMAPNPDDMEEDP) 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 PKCnu 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(ab')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.

Cell Culture and Transient Transfections-- Human Raji and Ramos and mouse A20 B-cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 units/ml penicillin/streptomycin, 2 mM glutamine, and 50 µM 2-beta -mercaptoethanol. Chicken DT40 cells were cultured in RPMI 1640 medium in the presence of 10% fetal bovine serum, 1% chicken serum, 10 units/ml penicillin/streptomycin, and 2 mM glutamine. HEK 293 endothelial cells expressing the TET Repressor protein were maintained in DMEM supplemented with 10% fetal bovine serum, 10 units/ml penicillin/streptomycin, 2 mM glutamine, and 5 µg/ml blasticidin.

Transient transfection of HEK 293 cells was carried out using a Beckman Gene-Pulser electroporation apparatus. 1 × 107 cells/0.5 ml serum-free media were pulsed in 0.4-cm cuvettes with 10 µg 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 PKCnu coding sequence was PCR-amplified from a human brain cDNA library (Clontech) using 5'-ACGTGCGGCCGCTGTCTGCAAATAATTCCCCTCCATCAGCCCAG-3' forward and 5'-ACGTTCTAGATTAAGGATCTTCTTCCATATCATCTGGATTAGG-3' 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-PKCnu protein. A similar method was used to construct the pcDNA4/TO FLAG-PKD vector. To generate the pcDNA4/TO GFP-PKCnu construct, the coding sequence for GFP was PCR-amplified with 5'-HindIII and 3'-NotI restriction sites. This PCR fragment was then cloned into the pcDNA4/TO vector, and the PKCnu coding sequence was cloned in-frame COOH-terminal to the GFP sequence using NotI and XbaI restriction sites. PKCalpha , PKCepsilon , and PKCtheta 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 PKCnu , resulting in single or double amino acid substitutions, were generated by overlap PCR using wild-type PKCnu 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: PKCnu -K605N, 5'-GGGAGGGATGTGGCTATTAACGTAATTGATAAGATGAG-3'; PKCnu -S731A/S735A, 5'-CATTGGTGAAAAGGCATTCAGGAGAGCTGTGGTAGGAACTCCAGC-3'; and PKCnu -S731E/S735E, 5'- CATTGGTGAAAAGGAATTCAGGAGAGAGGTGGTAGGAACTCCAGC-3'.

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 µg/ml doxycycline for 24 h.

Cell Lysis and Immunoprecipitation-- Cells were lysed in a buffer containing 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, protease inhibitors, 1 mM AEBSF, and 1% Triton X-100. Exogenously expressed PKCnu was immunoprecipitated with either a FLAG monoclonal antibody or with an affinity-purified PKCnu antibody recovered with protein G-Sepharose beads and resuspended in 2× SDS-PAGE reducing sample buffer (1 M Tris-HCl, pH 6.8, 0.1 mM Na3VO4, 6% SDS, 0.5 M EDTA, 4% 2-beta mercaptoethanol, 10% glycerol, 0.01% bromphenol blue). Immunoprecipitates were separated by 8% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and Western blotted with appropriate antibodies.

Cell Fractionation-- DT40 B-cells were washed in ice-cold phosphate-buffered 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 × g for 10 min at 4 °C. The supernatant was subjected to high speed ultracentrifugation at 100,000 × 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 × g for 10 min at 4 °C. PKCnu 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 MgCl2). PKCnu autophosphorylation was determined by incubating immunocomplexes with 20 µl of kinase buffer containing 100 µM [gamma -32P]ATP at 30 °C for 10 min. Reactions were terminated by the addition of 2× SDS-PAGE sample buffer, and the samples were analyzed by 8% SDS-PAGE and autoradiography.

Microscopy-- For immunofluorescent localization of endogenous PKCnu , 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-PKCnu , 1 µg/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-PKCnu 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although the PKCnu gene and transcripts have been described previously (17), there is no present literature regarding its regulation or receptor systems that utilize PKCnu as a signaling mechanism. However, PKCnu has two putative C1 domains. These domains (see schematic in Fig. 1A) of ~50 residues are thought to bind the lipid second messenger diacylglycerol, suggesting that PKCnu 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 PKCnu in DAG signaling by imaging the subcellular localization of PKCnu in cells that had been left untreated or treated with PMA (Fig. 1B). As can be seen, both endogenous PKCnu (imaged in fixed and antibody-stained cells, top panels) and GFP-tagged PKCnu (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 PKCnu .


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Fig. 1.   Activation of PKCnu by the DAG mimic PMA. A, schematic of PKCnu showing position of C1 domains, the single central pleckstrin homology (PH) domain, and the location of ATP binding site and putative activation loop serines. B, membrane trans-location of endogenous and GFP-tagged PKCnu by PMA. Top panels, trans-location of endogenous PKCnu . DT40 B-cells were left untreated or treated with PMA for 10 min, fixed, stained with an anti-PKCnu antibody, and imaged. Bottom panels, GFP-tagged PKCnu was expressed in A20 B-cells, and live cells were imaged during PMA stimulation. Images shown are before and 8 min after the addition of PMA. The homogenous staining pattern obtained upon imaging the GFP-tagged PKCnu in live cells suggests that the punctate staining pattern observed for endogenous PKCnu (both before and after PMA treatment) is an artifact of the fixation process. C, PMA induces activation and serine 735 phosphorylation of PKCnu . Left panel, expression of FLAG-tagged PKCnu . A FLAG-PKCn expression construct was transfected into HEK 293 cells under control of a doxycycline-responsive promoter. Cells were lysed, immunoprecipitated with the indicated antibody, and analyzed by anti-FLAG immunoblotting. Right panel, anti-pS735 antibody recognizes activated FLAG-PKD and FLAG-PKCnu . HEK 293 cells expressing either FLAG-PKD or FLAG-PKCnu were lysed, immunoprecipitated with anti-FLAG antibody, and analyzed by in vitro kinase assay, anti-pS735 immunoblotting, and anti-FLAG immunoblotting. D, PMA induces the activation of endogenous PKCnu in chicken and human B-cell lines. The indicated cell lines were treated or not treated with PMA, lysed, immunoprecipitated with anti-PKCnu , and analyzed by anti-pS735 and PKCnu immunoblotting.

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 PKCnu (18, 19). To understand how membrane recruitment affects PKCnu function, we produced a FLAG-tagged PKCnu construct as a backbone for mutational analysis of PKCnu activation and confirmed its expression after transfection of HEK 293 cells (Fig. 1C, left panel). The treatment of cells expressing FLAG-PKCnu 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 PKCnu 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-PKCnu . Anti-pS735 immunoreactivity strongly correlated with the activation state of FLAG-PKCnu , suggesting a role for phosphorylation of this residue during PKCnu activation (Fig. 1C, right panel, middle blot). We further utilized anti-pS735 to examine at the activation of endogenous PKCnu . As our initial analyses (data not shown) had indicated the presence of abundant PKCnu in several B-cell lines, we evaluated whether PMA treatment could activate endogenous PKCnu 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-PKCnu immunoprecipitates, indicating that endogenous PKCnu is activated by PMA in the same manner as the recombinant FLAG-PKCnu .

To further investigate the role of pS735 phosphorylation in PKCnu activation, we constructed a kinase-deficient mutant of PKCnu on the FLAG-PKCnu backbone via the mutation of a conserved lysine residue within the putative ATP-binding cassette of the kinase domain (FLAG-PKCnu -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 wild-type FLAG-PKCnu with that of the FLAG-PKCnu -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 PKCnu by producing mutants on the FLAG-PKCnu 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 PMA-independent, indicating that phosphorylation at serines 731 and 735 is both necessary and sufficient for PKCnu activation. When viewed in conjunction with the redistribution to the plasma membrane induced by PMA treatment, a compelling model for PKCnu activation can be constructed in which its activation occurs as the result of its membrane trans-location and subsequent trans-phosphorylation by an upstream PMA-regulated protein kinase on serine 731/735.


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Fig. 2.   Activation of PKCnu is via trans-phosphorylation of serines 731 and 735. A, PMA induces trans-phosphorylation of serine 735 of PKCnu . HEK 293 cells were transiently transfected with pcDNA4/TO vectors driving the expression of WT or PKCnu -KN mutant. 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 PKCnu autophosphorylation) or by anti-pS735 and anti-FLAG immunoblotting. Note that the anti-pS735 immunoreactivity together with the lack of kinase activity of the PKCnu -KN mutant demonstrates that a significant fraction of anti-pS735 immunoreactivity of stimulated PKCnu is because of a trans-phosphorylation event. B, phosphorylation of activation loop serines is necessary and sufficient for PKCnu 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.

Based on its abundance in B-cells, its activation by the DAG mimic PMA and the well established role of PLCgamma in B-cell antigen receptor signal transduction, we next investigated whether PKCnu was activated after B-cell antigen receptor engagement. The engagement of the BCR on either chicken DT40 B-cells (Fig. 3A) or human Raji and Ramos human B-cells (Fig. 3B) induced strong and rapid activation of PKCnu as assessed by the induction of anti-pS735 immunoreactivity. To address the question of where activated PKCnu is localized within the cell, fractionation experiments were preformed in both PMA and BCR-stimulated B cells. As illustrated in Fig. 3C, PKCnu trans-locates 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-PKCnu from the cytosol to the plasma membrane (see Fig. 1B). In addition, pS735 immunoblotting reveals that activated PKCnu is restricted to the membrane fraction of PMA-treated B-cells (Fig. 3C, upper panels). In contrast, a portion of PKCnu trans-locates to the membrane fraction of BCR-stimulated B-cells, and activated PKCnu is detectable in both the cytosolic and membrane fractions (Fig. 3C). Kinetic analysis indicates that PKCnu rapidly redistributes from the cytosol to the membrane compartment of B-cells in response to BCR ligation (within <30 s) and that activated PKCnu is found in cytosolic and membrane compartments both at early (<30 s) and late (>= 10 min) time points (data not shown).


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Fig. 3.   B-cell receptor engagement induces activation of endogenous PKCnu 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-PKCnu , and analyzed by anti-pS735 and PKCnu immunoblotting. B, human Raji and Ramos B-cell lines were treated or not treated with F(ab')2 fragments of anti-human IgG, lysed at the indicated times, immunoprecipitated with anti-PKCnu , and analyzed by anti-pS735 and PKCnu immunoblotting. C, chicken DT40 B-cells were left untreated (-) 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 PKCnu activity was analyzed by anti-pS735 and PKCnu immunoblotting.

That the activation of PKCnu by BCR ligation is entirely dependent on PLCgamma activation was demonstrated through the use of DT40 B-cell lines engineered to be deficient in individual components of the signaling cascade, leading to PLCgamma activation (Fig. 4A). Lyn-deficient DT40 cells have intact but delayed PLCgamma activation (20). They also exhibit relatively intact but delayed activation of PKCnu (peak activation occurs at >10 min as opposed to ~1 min in wild-type DT40 cells). This closely tracks the published time course of PLCgamma activation as measured by inositol phosphate turnover (20). In contrast, DT40 cell lines deficient in BLNK, BTK, and PLCgamma 2, each of which have completely abrogated PLCgamma activation (reviewed in Ref. 21), show completely abrogated PKCnu activation. Note that in addition PMA-mediated PKCnu 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 PKCnu activation. Consistent with these results (Fig. 4B), the treatment of cells with the putative DAG antagonist calphostin C also abrogated BCR-mediated PKCnu activation in chicken and human B-cells.


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Fig. 4.   BCR-activation of PKCnu is dependent on PLCgamma and DAG. A, all panels, wild-type and mutant DT40 B-cell lines lacking the indicated signaling molecules were analyzed for BCR-mediated activation of PKCnu . Cells were left unstimulated (-) or were stimulated with either 50 ng/ml PMA for 10 min (P) or with 10 µg/ml anti-chicken IgM (B) for the indicated times. Cells were lysed, and the endogenous PKCnu was immunoprecipitated (IP). Samples were analyzed by SDS-PAGE and Western blotting using the indicated antibodies. B, wild-type DT40 B-cells were left untreated or were treated with 3.5 µM of the competitive DAG antagonist calphostin C (Cal.C) prior to PMA (P) or BCR stimulation (times are indicated) and analysis of PKCnu activity as in A. DMSO, Me2SO.

The above results demonstrate that PLCgamma is a probable source of DAG for BCR-mediated PKCnu 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 PKCnu would be localized, we investigated whether either of these classes of enzymes might serve as an upstream activating kinase for PKCnu . Consistent with this possibility, the treatment of B-cells with the classical/novel PKC inhibitor Ro-31-8025 completely blocked BCR-mediated PKCnu 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 PKCnu (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 PKCnu , we tested the ability of activated mutants of various PKC subtypes to induce PKCnu phosphorylation (and thus activation) in a heterologous expression assay. The expression of constitutively activated mutants of novel PKC isozymes (eta , epsilon , and theta ) produced robust constitutive activation of PKCnu in the absence of PMA stimulation (Fig. 5B). In contrast, the co-expression of kinase-deficient or wild-type PKCepsilon or PKCtheta had little or no effect on basal or PMA-induced activation of PKCnu . Interestingly, the expression of a constitutively activated classical PKC enzyme, PKCalpha , produced no detectable change in either constitutive or PMA-induced PKCnu activation (Fig. 5C), suggesting that PKCnu is a poor substrate for PKCalpha and potentially other classical PKC isoforms.


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Fig. 5.   Novel PKC isoforms control the phosphorylation and thus activity of PKCnu in intact cells. A, the classical/novel PKC-specific inhibitor Ro-31-8425 blocks PKCnu activation. Wild-type DT40 B-cells were left untreated or were treated with 5 µM 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 PKCnu was immunoprecipitated (IP) and analyzed by Western blotting with the indicated antibodies. DMSO, Me2SO. B, top and middle panels, HEK 293 cells were co-transfected with pcDNA4/TO FLAG-PKCnu 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 PKCnu 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-PKCnu and a control vector, a dominant active PKCalpha construct, or a dominant active PKCeta expression construct as indicated. Cells were treated or not treated with PMA and lysed, and endogenous PKCnu was immunoprecipitated and analyzed by Western blotting with the indicated antibodies. Bottom panel, the expression of the PKCalpha DA mutant protein was confirmed by Western blotting with an anti-PKCalpha antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have analyzed the activation mechanism of the novel serine-threonine kinase PKCnu and show that PKCnu is activated by PMA and BCR-mediated DAG production via the trans-phosphorylation 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, PKCalpha , to induce constitutive PKCnu activation suggests that this trans-phosphorylation event may be mediated primarily by novel PKC enzymes. As PKCnu exhibits robust activation in response to BCR engagement, our results suggest that PKCnu is an important downstream target of activated novel PKC enzymes during BCR signaling.

The closest homologues of PKCnu are PKD1/PKCµ 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 PKCnu , PKD1 and PKD2 (similar to PKCnu ) appear to act downstream from both DAG and protein kinase C enzymes. Although the data in this paper suggest that PKCnu appears to relatively specifically targeted by novel PKC isoforms, PKD1 and PKD2 are activated by both classical and novel PKCs (19, 23-25). 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 calcium-regulated 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 PKCnu /PKD1/PKD2 substrates represent distinct signaling pathways downstream from DAG and PKCs.

Whether PKD1, PKD2, and PKCnu 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 PKCdelta in controlling the mechanisms of anergy and tolerance in B-cells. Because PKD1 and PKCnu are both expressed in B-cells and appear to be targets of novel PKC enzymes, either one or both could plausibly function as a link between PKCdelta (and possibly other novel PKC enzymes) and downstream effectors and mechanisms involved in the creation of B-cell energy and tolerance. Determining whether PKCnu 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.

    ACKNOWLEDGEMENT

We gratefully acknowledge Tomohiro Kurosaki for the Lyn, PLCgamma 2, BTK, and BLNK-deficient DT40 B-cell lines.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM45901 (to A. M. S).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.

§ To whom correspondence should be addressed: Dept. of Pediatrics, University of Washington and Children's Hospital and Regional Medical Center, 1959 N. E. Pacific Ave., Seattle, WA 98195. Tel.: 206-221-6446; Fax: 206-221-5469; E-mail: andrewms@u.washington.edu.

Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M211295200

    ABBREVIATIONS

The abbreviations used are: PLCgamma , phospholipase C isozyme gamma ; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PKD, protein kinase D; BCR, B-cell receptor; HEK, human embryonic kidney; GFP, green fluorescent protein; AEBSF, 4-(2'-aminoethyl)-benzenesulfonyl fluoride hydrochloride.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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