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• W2079742401 abstract "We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCα, PKCζ, PKCδ, and PKCμ. We have analyzed the interaction of PKCμ with p32 in detail, and we show here in vivo association of PKCμ, as revealed from yeast two-hybrid analysis, precipitation assays using glutathioneS-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKCμ is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKCμ in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKCμ, it does not serve as a substrate. Interestingly, PKCμ-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKCμ, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKCμ kinase activity. We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCα, PKCζ, PKCδ, and PKCμ. We have analyzed the interaction of PKCμ with p32 in detail, and we show here in vivo association of PKCμ, as revealed from yeast two-hybrid analysis, precipitation assays using glutathioneS-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKCμ is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKCμ in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKCμ, it does not serve as a substrate. Interestingly, PKCμ-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKCμ, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKCμ kinase activity. protein kinase C glutathione S-transferase polyacrylamide gel electrophoresis phosphate-buffered saline phorbol 12,13-dibutyrate c-Jun amino-terminal kinase The protein kinases C (PKC)1 comprise a family of intracellular serine/threonine-specific kinases, which are implicated in signal transduction of a wide range of biological responses including changes in cell morphology, proliferation, and differentiation (1Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1217) Google Scholar, 2Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4219) Google Scholar, 3Toker A. Front. Biosci. 1998; 3: 1134-1147Crossref PubMed Google Scholar). The currently defined 11 members of the PKC family can be grouped into the three major classes of Ca2+-dependent classical PKCs, Ca2+-independent, novel PKCs, and Ca2+- and lipid-independent atypical PKCs as well as PKCμ and its mouse homologue PKD (4Johannes F.J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 5Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar), which do not conform to either one of these major classes and may thus define a new subgroup (6Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (919) Google Scholar). PKCμ/PKD differ from the three major groups of PKC isozymes by the presence of an amino-terminal hydrophobic domain, an acidic domain (7Gschwendt M. Johannes F.J. Kittstein W. Marks F. J. Biol. Chem. 1997; 272: 20742-20746Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), a pleckstrin homology domain within the regulatory region (8Gibson T.J. Hyvonen M. Musacchio A. Saraste M. Birney E. Trends Biochem. Sci. 1994; 19: 349-353Abstract Full Text PDF PubMed Scopus (295) Google Scholar), and lack of a typical pseudosubstrate site. PKCμ is ubiquitously expressed, and evidence for the involvement of PKCμ in diverse cellular functions stems from reports showing enhancement of constitutive transport processes in PKCμ-overexpressing epithelial cells (9Prestle J. Pfizenmaier K. Brenner J. Johannes F.J. J. Cell Biol. 1996; 134: 1401-1410Crossref PubMed Scopus (100) Google Scholar), G protein-mediated regulation of Golgi organization (10Jamora C. Yamanouye N. Van Lint J. Laudenslager J. Vandenheede J.R. Faulkner D.J. Malhotra V. Cell. 1999; 98: 59-68Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), and involvement in protection from apoptosis (11Johannes F.J. Horn J. Link G. Haas E. Siemienski K. Wajant H. Pfizenmaier K. Eur. J. Biochem. 1998; 257: 47-54Crossref PubMed Scopus (64) Google Scholar). Interestingly, PKCμ shows particularly high expression in thymus and hematopoietic cells suggesting a potential role in immune functions (12Rennecke J. Johannes F.J. Richter K.H. Kittstein W. Marks F. Gschwendt M. Eur. J. Biochem. 1996; 242: 428-432Crossref PubMed Scopus (43) Google Scholar). In accordance with this is the finding that, upon B cell receptor stimulation, PKCμ is recruited together with the tyrosine kinase Syk and phospholipase Cγ to the B cell receptor complex and negatively regulates phospholipase Cγ activity (13Sidorenko S.P. Law C.L. Klaus S.J. Chandran K.A. Takata M. Kurosaki T. Clark E.A. Immunity. 1996; 5: 353-363Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In addition to lipid second messengers as regulators of PKC translocation and activation, there is increasing evidence for a role of regulatory proteins in controlling kinase activity and/or intracellular location of various PKC members. Thus, the identification of receptors of activated protein kinase C (14Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar) as well as the binding of more general and of specific modulators such as 14-3-3 (15Aitken A. Collinge D.B. van Heusden B.P. Isobe T. Roseboom P.H. Rosenfeld G. Soll J. Trends Biochem. Sci. 1992; 17: 498-501Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 16Burbelo P.D. Hall A. Curr. Biol. 1995; 5: 95-96Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), PAR4, LIP (18Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 19Diaz-Meco M.T. Municio M.M. Sanchez P. Lozano J. Moscat J. Mol. Cell. Biol. 1996; 16: 105-114Crossref PubMed Google Scholar), and ZIP (20Puls A. Schmidt S. Grawe F. Stabel S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6191-6196Crossref PubMed Scopus (193) Google Scholar), respectively, points to a complex regulation of PKC-dependent intracellular pathways. Whereas the latter selectively bind to the C1 regulatory domain of the atypical PKCλ and ζ and regulate kinase activity in a lipid messenger-independent manner (18Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), protein interacting with protein kinase Cs 1 was identified as a PKCα kinase domain-binding protein (21Staudinger J. Zhou J. Burgess R. Elledge S.J. Olson E.N. J. Cell Biol. 1995; 128: 263-271Crossref PubMed Scopus (261) Google Scholar). By analogy, because of the ubiquitous expression of PKCμ and its apparent involvement in diverse cellular responses, the existence of cell type- and/or organelle-specific regulators of PKCμ can be postulated. Indeed, 14-3-3 proteins as well as phosphatidylinositol 4-kinases were recently identified to be associated specifically with the C1 region of PKCμ (17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Nishikawa K. Toker A. Wong K. Marignani P.A. Johannes F.J. Cantley L.C. J. Biol. Chem. 1998; 273: 23126-23133Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To define other interacting proteins and to investigate their role in modulating kinase activity, we have used different PKCμ domains in various screening assays for PKCμ-binding proteins. The pleckstrin homology domain and the kinase domain of PKCμ were used in a yeast two-hybrid screen in order to identify new PKCμ-binding proteins. With the kinase domain as a bait, a novel PKC-binding protein was detected. We identified the multifunctional chaperon protein p32, previously described as a receptor of complement component C1q (23Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (314) Google Scholar), the kininogen-binding protein p33 (24Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), and splicing factor associated protein p32 (25Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-394Abstract Full Text PDF PubMed Scopus (411) Google Scholar) as a general PKC interactor, and we describe in detail its interaction with PKCμ and the functional consequences on kinase activity. To introduce BamHI restriction sites, the kinase domain of PKCμ covering amino acids 570–911 was amplified using the following primers: 5′-ATCCTCATAGGATCCAAATCACTA-3′ and 5′-ATCTCCTAGGATCCGTCAAAAC-3′. The amplified cDNA fragment was cloned either in pAS1 for yeast two-hybrid screening or in pGEX-3X (GST-μKin) to express glutathioneS-transferase (GST) fusion proteins. The yeast strain Y190 was transformed with pAS1/PKCμ according to standard procedures (26Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (366) Google Scholar). Expression of the fusion protein was verified by Western blot analysis of yeast lysates using a PKCμ-specific antibody. A pACT λ bacteriophage library of human-activated B-lymphocytes was convertedin vitro to plasmids (27Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (321) Google Scholar) and used to transform the pAS1/PKCμ-expressing Y190 yeast strain according to standard conditions (26Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (366) Google Scholar). Clones were selected on the respective medium lacking tryptophan, leucine, and histidine containing 50 mm3-amino-1,2,4-triazole (Sigma). Upon day 4 grown colonies were analyzed by lacZ staining. Blue colonies were streaked again and confirmed by lacZ staining. pACT plasmids were recovered by bacterial transformation of yeast isolated plasmids and subjected to dideoxy sequencing of both strands. The production and purification of PKCμ from Sf158 insect cells overexpressing PKCμ has been described (28Dieterich S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus (76) Google Scholar). To produce GST-p32 fusion proteins the cDNA fragment was amplified from the pACT-p32 plasmid using primers to introduce a BamHI site 5′ of the ATG and cloned in frame in pGEX-3X. The fusion proteins for precipitation analysis were prepared according to standard procedures. The construction of the GFP-p32 construct (29Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar) and the c-Myc-tagged PKCμ expression plasmid has been described previously (22Nishikawa K. Toker A. Wong K. Marignani P.A. Johannes F.J. Cantley L.C. J. Biol. Chem. 1998; 273: 23126-23133Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 28Dieterich S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus (76) Google Scholar). The human SKW 6.4 B cell line (ATCC) was cultured in RPMI medium supplemented with 5% fetal calf serum. 293T cells were obtained from ATCC. A rabbit antibody and a monoclonal antibody directed against gC1qR/p32 were used (29Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar, 30Ghebrehiwet B. Lu P.D. Zhang W. Lim B.L. Eggleton P. Leigh L.E. Reid K.B. Peerschke E.I. Hybridoma. 1996; 15: 333-342Crossref PubMed Scopus (63) Google Scholar). PKCμ, PKCζ, PKCδ, and JNK were detected with rabbit antibodies (Santa Cruz Biotechnology and Roche Molecular Biochemicals), PKCα and cytochromec with monoclonal antibodies (Santa Cruz Biotechnology; PharMingen), and GST with a goat antibody (Amersham Pharmacia Biotech). Secondary alkaline phosphatase-conjugated goat anti-mouse IgG + IgM, goat anti-rabbit IgG antibodies, Cy3-conjugated goat anti-mouse and Cy2-conjugated goat anti-mouse antibodies were purchased from Dianova. Protease and phosphatase inhibitors were from Biomol. Phorbol ester (phorbol 12,13-dibutyrate, PdBu) and phosphatidylserine were purchased from Sigma. SKW 6.4 and Sf158 cells were lysed at 4 °C in lysis buffer (20 mm Tris/HCl, pH 7.4, 1% Triton X-100, 150 mm NaCl, 5 mm EDTA, pH 7.4, 1 mmNaF, 1 mm NaPP, 1 mm sodium orthovanadate, 1 mm sodium molybdate, 1 mm p-nitrophenyl phosphate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride). After 60 min cell lysis the lysates were centrifuged (10,000 ×g, 15 min, 4 °C), and immunoprecipitation was performed as described (31Storz P. Doppler H. Wernig A. Pfizenmaier K. Muller G. FEBS Lett. 1998; 440: 41-45Crossref PubMed Scopus (30) Google Scholar). GST fusion protein pull-down assays were performed by incubation of 1 ml of lysate (representing 500,000 Sf158 cells or 50 × 106 SKW 6.4 cells) with the indicated amounts of GST fusion proteins coupled to glutathione-Sepharose for 60 min at 4 °C. Immunocomplexes or GST complexes were washed three times and applied to SDS-PAGE followed by transfer to nitrocellulose membrane. Western blot detection of PKCμ or p32 was performed according to standard procedures. 80 ng of purified recombinant PKCμ from Sf158 cells were preincubated as indicated with different amounts of GST or GST-p32 in phosphorylation buffer (50 mm Tris, pH 7.5, 10 mmMgCl2, 2 mm dithiothreitol) for 10 min at room temperature. Kinase reaction in the absence or presence of phosphatidylserine/PdBu micelles, with or without 5 μg of aldolase as substrate, was started by addition of 4 μCi of [γ-32P]ATP (Amersham Pharmacia Biotech) in 10 μl of kinase buffer, and incubation was carried out for 15 min at 37 °C. The reaction was stopped by adding 5× concentrated sample buffer, subsequently fractionated on 12% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed upon autoradiography. Autoradiographs were analyzed by quantitative PhosphorImager analysis (Molecular Dynamics). After immunoprecipitation PKCμ substrate and autophosphorylation were determined in in vitro kinase assays as described (17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). For cellular fractionation 4 × 108 SKW 6.4 and 3 × 108 293T cells were resuspended in lysis buffer containing no detergent and homogenized by applying 15 strokes with a “very tight-fitting” 5-ml Dounce homogenizer (Braun, Melsungen, Germany). Cellular debris was removed by centrifugation (800 × g, 5 min). The remaining lysate was centrifuged at 100,000 × g. The supernatant containing the cytosolic fraction was defined as the soluble fraction. The pellet was dissolved in lysis buffer containing 1% Triton X-100 and defined as the non-soluble fraction. Cells were washed twice with PBS and fixed in 3.5% paraformaldehyde (in PBS) for 15 min at 37 °C. Permeabilization and blocking of the cells proceeded through incubation with 0.05% Tween 20 and 5% normal goat serum in PBS for 30 min. The cells were rinsed 3 times with PBS and then incubated with primary antibodies (0.05% Tween 20 and 5% normal goat serum in PBS). For immunofluorescence detection of the indicated proteins (see Figs. 3 and 4), cells were simultaneously incubated for 2 h with two different antibodies used in the following concentrations: PKCμ antibody at 4 μg/ml, anti-p32 or anti-coatomer CM1-A10 monoclonal antibodies at 3 μg/ml, p24 antibody at 1 μ g/ml, and anti-cytochrome C monoclonal antibody at 1 μg/ml. Incubation with Cy3- and Cy2-conjugated secondary antibodies (2 μg/ml) was performed for 1 h. Following staining, the cells were rinsed four times with PBS and mounted in mounting medium from Sigma (PBS/glycerol). In control stainings, no cross-reactivity of the anti-mouse and anti-rabbit antibodies was observed (data not shown). A Leica confocal laser-scanning microscope was used for colocalization studies. Simultaneous excitation of fluorescent dyes was achieved by an argon/krypton laser. The following adjustments were made: 1) excitation filter, short pass KP590; 2) beam splitter module, neutral beam splitter; 3) channel 1 Cy3 emission, barrier filter long pass OG590; and channel 2 Cy2 emission, barrier filter band pass BP535. Images were acquired through a plan 100 or 63× (1.3 oil immersion) objective. The cells were sliced into horizontal optical sections at an interval of 1 μm.Figure 4PKC μ associates with p32 in mitochondria. SKW 6.4 cells were coimmunostained with antibodies against PKCμ, p32, and cytochromec. Cells proceeded to confocal laser scan analysis as described under “Materials and Methods.” p32 (red) colocalizes with cytochrome c (green) as indicated by the blue color in the overlay (top row, right panel). PKCμ (red) colocalizes partially with p32 (green) shown by the overlay (middle row, right panel). PKCμ (red) associates partially with cytochrome C (green) as shown by the blue color(bottom row, right panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cDNA fragment encoding for the PKCμ kinase domain was amplified by polymerase chain reaction, cloned in frame into pAS1 to be expressed as a fusion protein with the DNA binding domain of Gal4, and used in a two-hybrid screen in yeast (see scheme in Fig.1 A). After primary transfection of yeast and verification of PKCμ/Gal4 fusion protein expression by immunoblot using a PKCμ kinase domain-specific antibody (data not shown), yeast cells were secondarily transfected with pACT vector containing a human B cell library expressing fusion proteins with the Gal4 DNA activation domain. Transfectants were selected for growth on aminotriazole-containing minimal medium according to standard procedures (27Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (321) Google Scholar, 32Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar). Upon β-galactosidase staining nine blue colonies growing at 50 mm 3-amino-1,2,4-triazole were identified. Plasmids were retrieved and subjected to sequence analysis of both strands using pACT-based primers. With the exception of one clone (Clone 138) all retrieved pACT plasmids displayed nonsense sequences resulting in premature translation termination and did not reveal Gal4 activation domain fusion proteins of significant length. Fig.1 B shows growth of Clone 138 on YPEG medium and the respective selection medium. The pACT plasmid was subjected to complete sequence analysis revealing a 1.5-kilobase pair cDNA insert. Upon data base searching, the sequence showed identity to a previously published cDNA coding for the glycoprotein p32/gC1q-R which binds to the globular head of C1q (23Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (314) Google Scholar). In addition to the complete coding region of p32, 21 base pairs of the 5′-untranslated region were included in the pACT cDNA insert resulting in an in frame addition of seven amino acids between activation domain of Gal4 and p32. For an independent experimental verification of PKCμ-p32 interaction, precipitation assays with GST fusion proteins were performed. Therefore, the coding region of p32 was polymerase chain reaction-amplified, cloned in frame into pGEX-3X, and expressed as bacterial fusion protein with glutathione S-transferase. Purified GST-p32, immobilized on glutathione-Sepharose beads, was used to precipitate PKCμ from whole cell extracts of PKCμ-expressing Sf158 cells (Fig. 2 A, top left panel). PKCμ could be specifically detected by Western blot analysis in GST-p32 precipitates using as little as 1 μg of fusion protein, whereas no signal was revealed in control precipitates using up to 16 μg of GST protein (Fig. 2 A, left panels). GST 14-3-3τ, which efficiently associates with the PKCμ regulatory domain (17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), served as a positive control (Fig. 2 A, left panel, right lane). The respective amounts of fusion proteins used in precipitation assays were visualized by immunoblotting using an anti-GST antibody (Fig. 2 A, bottom left panels). The same result was obtained using purified recombinant PKCμ (Fig. 2 A, top right panel), indicating direct molecular interaction of PKCμ with p32. The reciprocal precipitation was carried out with extracts from the B cell line SKW 6.4, which expresses p32 in significant amounts (see Figs. 2 C and 4) and a purified GST fusion protein of the PKCμ kinase domain (GST-μKin). Precipitates were analyzed by immunoblotting using a p32-specific rabbit antibody (Fig. 2 B, top panel) or a GST-specific antibody to estimate GST loads (Fig. 2 B, bottom panels). To demonstrate the specificity of the association, excessive amounts of GST protein (10-fold over GST-μKin) served as a negative control, which resulted in only a very weak staining (Fig. 2 B, right lanes). Thus, the data presented here provide clear evidence of specific association of p32 with the kinase domain of PKCμ. In parallel to the GST-p32 precipitation assays (Fig. 2, Aand B), association between PKCμ and p32 was independently demonstrated by reciprocal coimmunoprecipitation analysis using p32- and PKCμ-specific antisera (Fig. 2 C). The somewhat weaker signal of p32 observed in PKCμ immunoprecipitates might be due to a steric hindrance by the PKCμ antibody, which is directed against carboxyl-terminal epitopes and thus could be in proximity to the p32 binding region. Phorbol ester treatment of cells or addition of phosphatidylserine/phorbol ester micelles to in vitropull-down assays did not enhance p32 binding to PKCμ (data not shown), suggesting a constitutive, lipid-independent association of p32 in SKW 6.4 cells. In order to assess the selectivity of p32 interaction with PKCμ, other PKC isotypes were analyzed by pull-down assays and coimmunoprecipitation analyses using three different recombinant PKC isotypes representing the three major PKC subgroups. By using GST-p32, in addition to PKCμ, a specific binding of PKCα, PKCζ, and PKCδ was noted (Fig. 3 A). As a control, precipitation analysis of the c-Jun amino-terminal kinase (JNK) from lysates of 293T cells was performed. As shown in Fig.3 A (bottom panel), no binding of JNK could be detected. These results indicate a PKC-selective association of p32. Coimmunoprecipitation analyses using PKC-specific antibodies further confirmed interaction of p32 with members of the different PKC subgroups (Fig. 3 B). The association of PKCμ and p32 was further analyzed by confocal laser scanning microscopy. The literature on the cellular distribution of p32 is controversial, reporting p32 either localized at the cell membrane (33Peterson K.L. Zhang W. Lu P.D. Keilbaugh S.A. Peerschke E.I. Ghebrehiwet B. Clin. Immunol. Immunopathol. 1997; 84: 17-26Crossref PubMed Scopus (54) Google Scholar), intracellularly (34van den Berg R.H. Prins F. Faber-Krol M.C. Lynch N.J. Schwaeble W. van Es L.A. Daha M.R. J. Immunol. 1997; 158: 3909-3916PubMed Google Scholar), or at mitochondria (29Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar), which may reflect cell-specific differences. Therefore, we investigated the intracellular localization of p32 in the SKW 6.4 B cell line. As shown in Fig. 4, in these cells p32 is localized predominantly at intracellular compartments (Fig. 4,top row, left panel). Costaining with antibodies against cytochrome c (Fig. 4, top row, middle panel) resulted in a nearly identical staining pattern, which was confirmed by overlay analysis indicated by the blue color shown in the top right panel. In SKW 6.4 cells, PKCμ shows a broad speckled distribution throughout extranuclear regions of the cell (Fig.4, middle row, left panel), with a clear enrichment in p32 positive, compartmentalized structures (Fig. 4, middle row, middle panel). Overlay of PKCμ- and p32-specific staining verifies a partial colocalization of both proteins (Fig. 4,middle row, right panel). A double staining with PKCμ (Fig. 4, bottom row, left panel) and cytochrome c(Fig. 4, bottom row, middle panel)-specific antibodies confirmed that PKCμ is partially located at mitochondria in SKW 6.4 cells (shown in blue at Fig. 4 in the bottom row, right panel). In 293T cells (Fig.5, upper panel) and in SKW 6.4 cells, only a weak colocalization signal with p24 was revealed (Fig. 5,bottom panels), which is in accordance with an enrichment of PKCμ at mitochondria in the latter cell line. The data presented here thus indicate a cell type-specific compartmentalization/enrichment of PKCμ either at mitochondria, in the B cell line SKW 6.4 (Fig. 4), or at Golgi structures in 293T cells (Fig. 5). Since in in vitro studies p32 specifically binds to PKCμ and appears to be constitutively associated with the kinase in the B cell line SKW 6.4, we investigated whether it affects PKCμ kinase activity in vitro. Incubation of PKCμ with GST-p32 led to a slight enhancement of autophosphorylation (Fig.6). We analyzed further whether substrate phosphorylation is affected by GST-p32 binding to PKCμ also. As shown in Fig. 6 A, phosphorylation of the well known in vitro substrate aldolase (7Gschwendt M. Johannes F.J. Kittstein W. Marks F. J. Biol. Chem. 1997; 272: 20742-20746Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) was significantly inhibited over a 10-fold range (0.1–1 μg of p32). PKCμ-mediated aldolase phosphorylation was not affected in the presence of 1 μg of GST protein (Fig. 5 B, right lane), indicating that inhibition of aldolase phosphorylation was not due to unspecific effects of the GST moiety. Quantitative analysis revealed, in the presence of 1 μg of GST-p32 an approximately 70% inhibition of aldolase phosphorylation (Fig. 7 A, right panel). These data suggest that p32 binding to the kinase domain restricts, probably by steric hindrance, the substrate access to PKCμ.Figure 7Compartment-specific inhibition of PKC μ substrate phosphorylation. PKCμ was immunoprecipitated (IP) from soluble and non-soluble fractionated 293T and SKW 6.4 cells using equal amounts of cell lysates and subjected to in vitro auto- and substrate phosphorylation (B). PKCμ and p32 expression was monitored in PKCμ immunoprecipitates by Western blot analysis (A). Data from one of three experiments performed with similar results are shown. p, particulate fraction; s, soluble fraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A dose-dependent inhibition of substrate phosphorylation could be further demonstrated with in vitro kinase assays of immunoprecipitates (Fig. 6 B). Addition of increasing amounts of p32 to PKCμ immunoprecipitates from 293T cells significantly inhibited aldolase phosphorylation, whereas autophosphorylation remained largely unaffected. In vitro inhibition of PKCμ substrate phosphorylation by p32 was not influenced by a concomitant phorbol ester activation (Fig. 6 C) pointing to distinct regulation mechanisms. In SKW 6.4 cells, endogenous PKCμ has been localized predominantly to particulate structures, yet a weak cytosolic staining suggested a broader distribution to other compartments as well (see Fig. 4). Likewise, in several cell types, PKCμ also appeared to be enriched in particulate structures (9Prestle J. Pfizenmaier K. Brenner J. Johannes F.J. J. Cell Biol. 1996; 134: 1401-1410Crossref PubMed Scopus (100) Google Scholar), but a partial cytosolic location has also been reported (36Matthews S. Iglesias T. Cantrell D. Rozengurt E. FEBS Lett. 1999; 457: 515-521Crossref PubMed Scopus (70) Google Scholar) and can be observed in the cell lines analyzed here (Figs. 4 and 5). In order to investigate whether or not the p32-PKCμ interaction and regulation of kinase activity is restricted to specific intracellular compartments, cell fractionation experiments were performed with 293T cells and SKW 6.4 cells. The results obtained support a compartment-specific regulation of PKCμ kinase activity by p32. Both cell lines express similar levels of endogenous p32 (Fig.7 A, bottom panel). PKCμ immunoprecipitates from soluble and particulate fractions of both SKW 6.4 and 293T cells were analyzed using PKCμ-specific antibodies. As shown in Fig. 7 A, approximately equal amounts of PKCμ were present in either the soluble or the particulate fraction, yet p32 could be predominantly detected in PKCμ precipitates from the particulate fraction (Fig.7 A, middle panel), although, under the experimental conditions applied here, both fractions contain comparable amounts of p32 (Fig. 7 A, bottom panel). PKCμ immunoprecipitates were subjected to in vitroautophosphorylation and substrate phosphorylation. As shown in Fig.7 B, aldolase phosphorylation by PKCμ immunoprecipitates from the soluble fraction could be readily discerned, whereas PKCμ isolated from the particulate, p32-positive fraction did not show any detectable aldolase phosphorylation. Together with the data fromin vitro kinase assays with purified PKCμ and p32-GST fusion proteins (Fig. 6), these findings indicate a p32-dependent regulation of compartmentalized PKCμ kinase activity and suggest a new mechanism of regulation of kinase activity via kinase domain interacting proteins, identifying an as yet unrecognized functional role of p32 in this process. In this study, we identified by yeast two-hybrid screening a novel PKCμ-interacting protein, the previously described protein p32 (Fig.1), which has been associated with multiple, chaperon-like functions. p32 may serve as a compartment-specific regulator of PKCμ kinase activity. Cellular colocalization of PKCμ and p32 at mitochondria was shown in the B cell line SKW 6.4 by confocal immunofluorescence microscopy (Fig. 4). Functional interaction of both proteins was shown by precipitation analysis with GST fusion proteins as well as by coimmunoprecipitation indicating a constitutive association of p32 with PKCμ (Fig. 2). As p32 causes inhibition of PKCμ substrate phosphorylation (Figs. 6 and 7), we propose a novel model of a chaperon-mediated control of PKCμ activation, in which PKCμ function is restricted to defined cellular compartments by the multifunctional protein p32. Accordingly, PKCμ kinase regulation by p32 may not only serve as a paradigm to explain a differential, cell-, and/or compartment-specific activation of ubiquitously expressed kinases by virtue of a cell-specific intracellular location/function of regulatory molecules but also provides new insight into regulation of kinase activity toward specific substrates by kinase domain interacting proteins (Figs. 6 and 7). These data show that PKCμ is, in addition to its regulation by lipids and 14-3-3 proteins (17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), controlled by a p32-dependent mechanism that probably controls substrate access by steric hindrance. So far, the biological role of p32 appeared rather unclear due to the diverse functions reported; p32 has been originally identified as a cell surface protein binding to the globular “heads” of the complement factor C1q (23Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (314) Google Scholar). It also has been described as a cell surface kininogen-binding protein (24Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In addition, several independent reports have described p32 as an intracellular protein (35Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar,37Dedio J. Muller-Esterl W. FEBS Lett. 1996; 399: 255-258Crossref PubMed Scopus (50) Google Scholar), which colocalizes in the endothelial cell line EA.hy926 with a mitochondrial marker protein (29Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar). p32 has been shown to be important for the maintenance of mitochondrial oxidative phosphorylation (38Muta T. Kang D. Kitajima S. Fujiwara T. Hamasaki N. J. Biol. Chem. 1997; 272: 24363-24370Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Mitochondrial functions of p32 are further indicated by the identification of a yeast homologue of p32, called Mam33p, that has been localized to the inner mitochondrial membrane (39Seytter T. Lottspeich F. Neupert W. Schwarz E. Yeast. 1998; 14: 303-310Crossref PubMed Scopus (52) Google Scholar). Other reports confirmed that p32 is located at mitochondria, but in addition a nuclear localization was found and a function of p32 as part of an import machinery was postulated (40Matthews D.A. Russell W.C. J. Gen. Virol. 1998; 79: 1677-1685Crossref PubMed Scopus (158) Google Scholar). Moreover, p32 was described as part of the RNA splicing complex SF2 in HeLa cells (25Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-394Abstract Full Text PDF PubMed Scopus (411) Google Scholar). p32 has further been shown to associate with many viral proteins including HIV-1 Tat (41Yu L. Zhang Z. Loewenstein P.M. Desai K. Tang Q. Mao D. Symington J.S. Green M. J. Virol. 1995; 69: 3007-3016Crossref PubMed Google Scholar) and Rev (42Luo Y., Yu, H. Peterlin B.M. J. Virol. 1994; 68: 3850-3856Crossref PubMed Google Scholar) as well as with EBNA-1 of Epstein-Barr virus (43Wang Y. Finan J.E. Middeldorp J.M. Hayward S.D. Virology. 1997; 236: 18-29Crossref PubMed Scopus (136) Google Scholar). The latter p32 functions are all considered to modulate transcription factor activity. The participation in different biological processes like mitochondrial functions, transcription- and splicing factor modulation, and potential role in complement cascade or blood coagulation (44Peerschke E.I. Jesty J. Reid K.B. Ghebrehiwet B. Blood Coagul. & Fibrinolysis. 1998; 9: 29-37Crossref PubMed Scopus (18) Google Scholar) suggest a typical chaperon function of p32. In this paper, we describe a novel aspect of p32 biology with a functional role as an inhibitor of kinase activity. The presented data show that p32 binds to the kinase domain of PKCμ and, without being a substrate, inhibits phosphorylation of aldolase, yet maintains or even enhances the level of autophosphorylation. As different phosphorylation sites trigger the activation state of PKC isoforms (45Dutil E.M. Keranen L.M. DePaoli-Roach A.A. Newton A.C. J. Biol. Chem. 1994; 269: 29359-29362Abstract Full Text PDF PubMed Google Scholar, 46Van Lint J.V. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), similar mechanisms are conceivable for PKCμ. For the p32-mediated regulation of PKCμ activity, several possibilities may be considered. First, p32 may interfere with substrate phosphorylation by steric hindrance. Second, p32 binding to PKCμ could induce a conformational change such that endogenous autophosphorylation sites are preferentially used over cellular substrates. Third, the phosphorylation sites critical for kinase activation are blocked by p32, disabling substrate phosphorylation, yet leaving autophosphorylation at other serine residues of PKCμ unaffected. Several phosphorylation sites important for PKCμ activation have now been mapped within the catalytic domain (47Vertommen D. Rider M. Ni Y. Waelkens E. Merlevede W. Vandenheede J.R. Van Lint J. J. Biol. Chem. 2000; 275: 19567-19576Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), which is in accordance with the latter model of p32 interference with PKCμ function. Together, our data presented here thus indicate that, besides regulation of PKCμ kinase activity via the C1 domain either by activating lipid second messengers and phorbol ester (1Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1217) Google Scholar, 3Toker A. Front. Biosci. 1998; 3: 1134-1147Crossref PubMed Google Scholar) or inactivating 14-3-3 proteins (17Hausser A. Storz P. Link G. Stoll H. Liu Y.C. Altman A. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 1999; 274: 9258-9264Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), other domains are involved in modulating PKCμ activity also. Since in contrast to 14-3-3, p32 does not affect lipid-induced PKCμ autophosphorylation (Fig. 6), we propose that PKCμ activity is controlled by at least two independent mechanisms. Moreover, our finding that p32 binds to different PKC isoforms points to a more general p32-based mechanism of controlling PKC kinase activity. The differential cellular localization of p32 in different cell types (29Dedio J. Jahnen-Dechent W. Bachmann M. Muller-Esterl W. J. Immunol. 1998; 160: 3534-3542PubMed Google Scholar, 40Matthews D.A. Russell W.C. J. Gen. Virol. 1998; 79: 1677-1685Crossref PubMed Scopus (158) Google Scholar) may contribute to the compartment-specific functional role of various PKC isotypes, including PKCμ. As shown here, in the SKW 6.4 cell line, p32 largely colocalized with cytochrome c, indicative of a mitochondrial localization (Fig. 4). In full accordance with the in vitro binding studies, PKCμ partially colocalized with p32 at mitochondria, as revealed from confocal microscopy (Fig. 4) and cell fractionation studies (Fig. 7). Therefore, we propose that p32 is part of an intracellular receptor that retains PKCμ at intracellular compartments such as mitochondria and serves as a regulator of its kinase activity. We thank Stephen Elledge, Houston, TX, for the generous gift of the human B cell library and the two-hybrid reagents. We thank Felix Wieland (Heidelberg, Germany) and Kai Sohn (Stuttgart, Germany) for providing us with the p24 antibody. We also thank Heike Döppler for expert technical assistance in preparing the confocal images." @default.
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