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• W2059335098 abstract "In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) as a second messenger, we studied the activation of protein kinase Cα by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase Cα, we have determined and characterized the PtdIns(4,5)P2-binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysine-rich cluster located in the β3- and β4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P2; and the second, the already well described site formed by the Ca2+-binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase Cα needs Ca2+ before the PtdIns(4,5)P2-dependent activation of the enzyme can occur. In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) as a second messenger, we studied the activation of protein kinase Cα by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase Cα, we have determined and characterized the PtdIns(4,5)P2-binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysine-rich cluster located in the β3- and β4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P2; and the second, the already well described site formed by the Ca2+-binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase Cα needs Ca2+ before the PtdIns(4,5)P2-dependent activation of the enzyme can occur. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 1The abbreviations used are: PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; GST, glutathione S-transferase; HA, hemagglutinin; PA, phosphatidic acid; PKC, protein kinase C; PS, phosphatidylserine; PMA, 12-myristate 13-acetate; ENTH, epsin NH2-terminal; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPS, 1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphoserine; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; PDB, protein data bank; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; CALM, clathrin assembly lymphoid myeloid leukemia protein plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected in the plasma membrane but is also found in secretory vesicles, lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus (1Divecha N. Banfic H. Irvine R.F. EMBO J. 1991; 10: 3207-3214Crossref PubMed Scopus (415) Google Scholar, 2Tran D. Gascard P. Berthon B. Fukami K. Takenawa T. Giraud F. Claret M. Cell. Signal. 1993; 5: 565-581Crossref PubMed Scopus (52) Google Scholar, 3Martin T.F.J. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (452) Google Scholar, 4Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar, 5Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). PtdIns(4,5)P2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity, or it can act as a precursor for the generation of different second messengers. For example, several families of phospholipase C enzymes are responsible for the hydrolysis of PtdIns(4,5)P2in cells, leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (4Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (245) Google Scholar, 6Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar), which may, in turn, lead to the activation of different proteins such as some PKC isotypes. Protein kinase C (PKC) composes a large family of serine/threonine kinases, which is activated by many extracellular signals and plays a critical role in many signal-transducing pathways in the cell (7Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4232) Google Scholar, 8Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 9Toker A. Front. Biosci. 1998; 3: D1134-D1147Crossref PubMed Google Scholar). Based on their enzymatic properties, the mammalian PKC isotypes have been grouped into smaller subfamilies. The first group, which includes the classical isoforms α, βI, βII, and γ, can be distinguished from the other groups because its activity is regulated by diacylglycerol (DAG) and, cooperatively, by Ca2+ and acidic phospholipids, particularly phosphatidylserine (PS). Members of the second group are the novel mammalian (δ, ε, η, and θ) and yeast PKCs that are not regulated by Ca2+. The third group comprises the atypical PKC isoforms, ζ, ι, and λ, whose regulation has not been clearly established, although it is clear that they are not regulated by DAG or Ca2+ (8Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 10Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (851) Google Scholar). In classical PKC isoenzymes, Ca2+-dependent binding to membranes shows a high specificity for 1,2-sn-phosphatidyl-l-serine (11Lee M.H. Bell R.M. Biochemistry. 1991; 30: 1041-1049Crossref PubMed Scopus (71) Google Scholar, 12Newton A.C. Keranen L.M. Biochemistry. 1994; 33: 6651-6658Crossref PubMed Scopus (122) Google Scholar, 13Johnson J.E. Zimmerman M.L. Daleke D.L. Newton A.C. Biochemistry. 1998; 37: 12020-12025Crossref PubMed Scopus (41) Google Scholar, 14Conesa-Zamora P. Lopez-Andreo M.J. Gómez-Fernández J.C. Corbalán-Garcı́a S. Biochemistry. 2001; 40: 13898-13905Crossref PubMed Scopus (56) Google Scholar). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15Newton A. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (244) Google Scholar, 16Toker A. Mol. Pharmacol. 2000; 57: 652-658Crossref PubMed Scopus (284) Google Scholar) and to a variety of amphipathic membrane compounds, such as arachidonic acid and free fatty acids (17Khan W.A. Blobe G.C. Hannun Y.A. Cell. Signal. 1995; 7: 171-184Crossref PubMed Scopus (219) Google Scholar). Furthermore, in vitro experiments have demonstrated that the presence of other anionic phospholipids in the vesicles decreases the requirement of phosphatidylserine, suggesting that PKC activation, other than the classical activation pathway (activation of phospholipase C and production of diacylglycerol by hydrolysis of PtdIns(4,5)P2and increase of intracytosolic Ca2+), could take placein vivo (15Newton A. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (244) Google Scholar). In view of the interest in PtdIns(4,5)P2 as a second messenger, several studies (11Lee M.H. Bell R.M. Biochemistry. 1991; 30: 1041-1049Crossref PubMed Scopus (71) Google Scholar, 18Chauhan V.P. Brockerhoff H. Biochem. Biophys. Res. Commun. 1988; 155: 18-23Crossref PubMed Scopus (48) Google Scholar, 19Singh S.S. Chauhan A. Brockerhoff H. Chauhan V.P. Biochem. Biophys. Res. Commun. 1993; 195: 104-112Crossref PubMed Scopus (68) Google Scholar, 20Kochs G. Hummel R. Fiebich B. Sarre T.F. Marme D. Hug H. Biochem. J. 1993; 291: 627-633Crossref PubMed Scopus (36) Google Scholar, 21Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 22Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar, 23Palmer R.H. Dekker L.V. Woscholski R. Le Good J.A. Gigg R. Parker P. J. Biol. Chem. 1995; 270: 22412-22416Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 24Oh E.S. Woods A. Lim S.T. Theibert A.W. Couchman J.R. J. Biol. Chem. 1998; 273: 10624-10629Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) have addressed the activation of PKC isotypes by this class of lipid. However, the results show little consistency as to which of the different PKC isotypes are activated or as regards their specificity for the different lipids employed. These conflicting results are probably due to the different ways used by investigators to activate the enzyme. It has been described that the C2 domains of several proteins, such as synaptotagmin (25Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 26Schiavo G. Gu Q.M. Prestwich G.D. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13327-13332Crossref PubMed Scopus (261) Google Scholar, 27Zhang X. Rizo J. Sudhof T.C. Biochemistry. 1998; 37: 12395-12403Crossref PubMed Scopus (167) Google Scholar, 28Davletov B. Perisic O. Williams R.L. J. Biol. Chem. 1998; 273: 19093-19096Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Sutton R.B. Ernst J.A. Brunger A.T. J. Cell Biol. 1999; 147: 589-598Crossref PubMed Scopus (158) Google Scholar) and rabphilin 3A (30Chung S.H. Song W.J. Kim K. Bednarski J.J. Chen J. Prestwich G.D. Holz R.W. J. Biol. Chem. 1998; 273: 10240-10248Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) among others, bind PtdIns(4,5)P2 through co-linear sequences that consist of highly basic amino acidic residues (3Martin T.F.J. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (452) Google Scholar, 25Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 26Schiavo G. Gu Q.M. Prestwich G.D. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13327-13332Crossref PubMed Scopus (261) Google Scholar, 30Chung S.H. Song W.J. Kim K. Bednarski J.J. Chen J. Prestwich G.D. Holz R.W. J. Biol. Chem. 1998; 273: 10240-10248Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 31MacLaughling S. Wang J. Gambhir A. Murray D. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (698) Google Scholar). These lysine-rich sequences probably represent the inositol phosphate-binding portions of larger phosphoinositide-binding domains. For example, in synaptotagmin, this basic sequence is flanked by regions rich in hydrophobic residues that could mediate acyl chain interactions (3Martin T.F.J. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (452) Google Scholar). When we look at the amino acidic sequence of the PKCα-C2 domain, we observe that some of the Lys/Arg residues described for synaptotagmin are conserved. Previous studies in our laboratory (32Ochoa W.F. Corbalan-Garcia S. Eritja R. Rodriguez-Alfaro J.A. Gomez-Fernandez J.C. Fita I. Verdaguer N. J. Mol. Biol. 2002; 320: 277-291Crossref PubMed Scopus (69) Google Scholar) have suggested that the highly positive charged β3-β4-sheets could interact electrostatically with the negatively charged phospholipids located at the membrane surface. Whether or not this is significant in the context of the full-length protein is still not clear. In this paper, we focus on the characterization of the interaction mechanism between PKCα-C2 domain and PtdIns(4,5)P2 and the consequent enzyme activation. For this purpose, we cloned the PKCα C2 domain fused to glutathioneS-transferase (GST) and full-length PKCα fused to a hemagglutinin (HA) tag, which enabled us to perform binding and specific activity studies. Site-directed mutagenesis of key residues located in two areas of the C2 domain shed light on the interaction of the classical isoenzyme with PtdIns(4,5)P2 and its activation mechanism. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2) were purchased from Avanti Polar Lipids Inc. (Birmingham, AL). Phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3-phosphate were purchased from Echelon Biosciences Inc. (Salt Lake City, UT). Phosphatidylinositol and phosphatidylglycerol were purchased from Lipid Products (Nutfield, Surrey, UK), and phorbol 12-myristate 13-acetate (PMA) was from Sigma. Rat PKCα cDNA was a gift from Drs. Nishizuka and Ono (Kobe University, Kobe, Japan). The cDNA fragment corresponding to residues 158–285 of the PKCα-C2 domain and mutants was amplified using PCR (33Corbalán-Garcı́a S. Rodrı́guez-Alfaro J.A. Gómez-Fernández J.C. Biochem. J. 1999; 337: 513-521Crossref PubMed Scopus (60) Google Scholar). Full-length PKCα mutants were generated by PCR site-directed mutagenesis (14Conesa-Zamora P. Lopez-Andreo M.J. Gómez-Fernández J.C. Corbalán-Garcı́a S. Biochemistry. 2001; 40: 13898-13905Crossref PubMed Scopus (56) Google Scholar, 34Conesa-Zamora P. Gomez-Fernandez J.C. Corbalan-Garcia S. Biochim. Biophys. Acta. 2000; 1487: 246-254Crossref PubMed Scopus (30) Google Scholar). All constructs, both wild-type and mutant genes, were subcloned into the mammalian expression vector pCGN (a gift from Dr. Tanaka, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). This vector contains the cytomegalovirus promoter and the multicloning sites that allow expression of the genes fused 3′ to the HA epitope (35Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). All constructs were confirmed by DNA sequencing. The cDNA fragment corresponding to residues 158–285 of the PKCα-C2 domain and mutants was amplified using PCR (33Corbalán-Garcı́a S. Rodrı́guez-Alfaro J.A. Gómez-Fernández J.C. Biochem. J. 1999; 337: 513-521Crossref PubMed Scopus (60) Google Scholar). The pGEX-KG plasmid containing the PKCα-C2 domain was transformed into HB101 Escherichia coli cells. Proteins were expressed and purified as described in a previous work (33Corbalán-Garcı́a S. Rodrı́guez-Alfaro J.A. Gómez-Fernández J.C. Biochem. J. 1999; 337: 513-521Crossref PubMed Scopus (60) Google Scholar). HEK293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Transfection was performed with the Ca2+ phosphate method described by Wigler et al. (36Wigler M. Silverstein S. Lee L.S. Pellicer A. Cheng V.C. Axel R. Cell. 1977; 11: 223-227Abstract Full Text PDF PubMed Scopus (845) Google Scholar). Protein purification was performed as described by Conesa-Zamoraet al. (14Conesa-Zamora P. Lopez-Andreo M.J. Gómez-Fernández J.C. Corbalán-Garcı́a S. Biochemistry. 2001; 40: 13898-13905Crossref PubMed Scopus (56) Google Scholar). The procedure described by Davletov and Sudhof (37Davletof B. Sudhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar) was used with minor modifications. A total of 10 μg of PKCα-C2 domain bound to glutathione-Sepharose beads was used. Lipid vesicles were generated by mixing chloroform lipid solutions in the desired proportions and then dried from the organic solvent under a nitrogen stream and further dried under vacuum for 60 min. 1,2-Dipalmitoyl-l-3-phosphatidyl-N-[methyl-3H]choline (PerkinElmer Life Sciences; specific activity 56 Ci/mmol) was included in the lipid mixture as a tracer, a concentration of ∼3000–6000 cpm/mg phospholipid. Dried phospholipids were resuspended in buffer containing 50 mm Hepes, pH 7.2, 0.1 m NaCl, and 0.5 mm EGTA by vigorous vortexing and subjected to direct probe sonication (four cycles of 1 min). Beads with protein bound to them were prewashed with the respective test solutions and resuspended in 50 μl of the corresponding lipid solution. The mixture was incubated at room temperature for 15 min with vigorous shaking and then briefly centrifuged in a tabletop centrifuge. The beads were washed twice with 0.4 ml of the incubation buffer without liposomes. Liposome binding was then quantified by liquid scintillation counting of the beads. The data in the paper and figures are expressed in number of nmol of lipid bound/10 μg of protein. PKCα was incubated with multilamellar vesicles (500 μm total lipid in 0.15 ml) in a buffer containing 20 mm Tris/HCl, pH 7.5, 5 mm MgCl2, and 0.5 mm EGTA or 0.2 mm CaCl2 at room temperature for 15 min. Vesicle-bound enzyme was separated from the free enzyme by centrifuging the mixture at 13,000 × g for 30 min at 20 °C. Aliquots from the supernatants and pellets were separated by SDS-PAGE (12.5% separating gel). The proteins were transferred onto a nitrocellulose membrane after electrophoresis. Immunoblot analysis of the epitope tag fused to the protein was performed by using anti-HA antibody 12CA5 and developed with chemiluminescence reagents (PerkinElmer Life Sciences). The proteins were analyzed by densitometry. The lipids used for the reaction were previously dried under a stream of N2, and the last traces of organic solvent were removed by keeping the samples under vacuum for 1 h. Lipids were suspended in 20 mm Tris-HCl, pH 7.5, 0.05 mmEGTA and vortexed vigorously to form multilamellar vesicles. A 20-μl sample of the lipids was added to the reaction mixture (final volume, 150 μl), which contained 20 mm Tris-HCl, pH 7.5, 0.2 mg/ml histone III-S, 20 μm [γ-32P]ATP (300,000 cpm/nmol), 5 mm MgCl2, and 200 μm CaCl2. The reaction was started by addition of 5 μl of the PKCα purified from transfected HEK293 cells. After 10 or 30 min, the reaction was stopped with 1 ml of ice-cold 25% trichloroacetic acid and 1 ml of ice-cold 0.05% bovine serum albumin. After precipitation on ice for 30 min, the protein precipitate was collected on a 2.5-cm glass fiber filter (Sartorius) and washed with 10 ml of ice-cold 10% trichloroacetic acid. The amount of 32Pi incorporated into histone was measured by liquid scintillation counting. The linearity of the assay was confirmed from the time course of histone phosphorylation during 30 min. Additional control experiments were performed with mock cell lysates to estimate the endogenous PKCα and nonspecific activities, which represented less than 1% of the total enzyme activity measured. The PDB identifiers for the experimentally determined C2 and ENTH domain structures used in the calculations were 1DSY (38Verdaguer N. Corbalán-Garcı́a S. Ochoa W.F. Fita I. Gómez- Fernández J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (287) Google Scholar) and 1HFA(39Ford M.G. Pearse B.M.F. Higgins M. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Science. 2001; 291: 1051-1055Crossref PubMed Scopus (606) Google Scholar). The Swiss-PDB Viewer 3.7 program by GlaxoSmithKline R&D Geneva (57Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar) was used to visualize the structures. Our first aim was to characterize the biochemical properties of the interaction between PtdIns(4,5)P2 and the C2 domain of PKCα. For this, a recombinant fusion protein was used, in which the C2 domain of PKCα was NH2-terminally fused to GST (PKCα-C2 domain) (33Corbalán-Garcı́a S. Rodrı́guez-Alfaro J.A. Gómez-Fernández J.C. Biochem. J. 1999; 337: 513-521Crossref PubMed Scopus (60) Google Scholar). The dependence of phospholipid binding on the PtdIns(4,5)P2concentration was studied by using phospholipid vesicles containing POPC/POPS (4:1, mol/mol) in the presence and in the absence of 100 μm CaCl2 (Fig. 1 A). The results show that in the presence of Ca2+, PtdIns(4,5)P2 slightly inhibited the PKCα-C2 domain phospholipid binding activity, which amounted to 8 nmol regardless of the PtdIns(4,5)P2concentration in the lipid vesicles. More interesting were the results obtained in the absence of Ca2+ because, in this case, the PKCα-C2 domain bound to a similar phospholipid vesicle composition in a PtdIns(4,5)P2-dependent manner, showing a maximal binding of 7 nmol and a [PIP2] 12 value of 1 mol % (Fig. 1 A). These results suggest that the C2 domain of PKCα may interact with PtdIns(4,5)P2independently of Ca2+. To explore whether this mechanism is specific to PtdIns(4,5)P2 or is related to the net negative charges present in the membrane, binding assays were performed by substituting PtdIns(4,5)P2 as supplier of net negative charges to the phospholipid vesicles (POPC/POPS/PIP2, 75:20:5 mol/mol) for POPS (POPC/POPS, 70:30). It was observed that 7 nmol of lipid were bound in the former case and only 1.3 nmol in the latter case (Fig. 1 A, inset), suggesting that there is a certain specificity for PtdIns(4,5)P2 and that the increased binding observed under these conditions is not only dependent on the net negative charges present at the membrane vesicles. In order to study whether Ca2+ can affect the PKCα-C2 domain binding affinity in the absence of POPS in the lipid vesicles, binding assays were performed with POPC and increasing PtdIns(4,5)P2 concentrations in the absence and in the presence of 100 μm CaCl2 (Fig. 1 B). In the absence of Ca2+, lipid binding reached a maximum of 7 nmol and [PIP2] 12 = 1.2 mol %, whereas in the presence of Ca2+, binding reached 9 nmol and [PIP2] 12 = 0.3 mol %. This demonstrates that in the absence of POPS, Ca2+ slightly increases both the affinity of the protein for PtdIns(4,5)P2-containing vesicles and the maximal binding capacity. The data described above might be explained by the existence of two types of binding mechanism, one Ca2+/PS-dependent and the other mostly Ca2+-independent and PtdIns(4,5)P2-dependent. Whether these two mechanisms corresponded to two different sites in the domain or to just one site with different biochemical behaviors could still not be answered at this point. To address this question, we made use of two PKCα-C2 domain mutants. One of them (PKCα-C2-D246N/D248N) has been demonstrated to be affected in the Ca2+-binding site (33Corbalán-Garcı́a S. Rodrı́guez-Alfaro J.A. Gómez-Fernández J.C. Biochem. J. 1999; 337: 513-521Crossref PubMed Scopus (60) Google Scholar, 34Conesa-Zamora P. Gomez-Fernandez J.C. Corbalan-Garcia S. Biochim. Biophys. Acta. 2000; 1487: 246-254Crossref PubMed Scopus (30) Google Scholar) (Fig. 2 A) and does not bind Ca2+ or PS. The second (PKCα-C2-K209A/K211A) has been demonstrated to be affected in an area corresponding to a lysine-rich cluster located in the β3-β4-sheets (Fig. 2 C) and partially lacks its ability to bind PS or phosphatidic acid in the absence of Ca2+ (32Ochoa W.F. Corbalan-Garcia S. Eritja R. Rodriguez-Alfaro J.A. Gomez-Fernandez J.C. Fita I. Verdaguer N. J. Mol. Biol. 2002; 320: 277-291Crossref PubMed Scopus (69) Google Scholar). Fig. 2 B shows the results obtained when PKCα-C2-D246N/D248N mutant was employed in the binding assays using vesicles containing POPC and increasing concentrations of PtdIns(4,5)P2 in the absence of Ca2+. In this case, maximal binding activity was 8 nmol of lipid and [PIP2] 12 = 0.5 mol %. These results are slightly higher than those obtained when the wild-type C2 domain was used in the assay (Figs. 1 B and 2 B, dotted line), suggesting that the substitution of these residues located at the Ca2+-binding site has no effect on the ability of the domain to bind to PtdIns(4,5)P2-containing vesicles in a Ca2+-independent manner. Furthermore, the results obtained suggest that the neutralization of the Ca2+-binding sites performed by the mutagenesis strategy probably helps to reduce the negative electrostatic potential exhibited by this area, in a similar way to the effect brought about by Ca2+ when it binds to this region (40Murray D. Honig B. Mol. Cell. 2002; 9: 145-154Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), thus facilitating the slight increase in the binding affinity observed in both cases. When the second mutant, PKCα-C2-K209A/K211A, was included in the binding assay under the same conditions as described above (Fig. 2 D), only 1.3 nmol of lipids were bound to the protein, indicating that these two residues are directly involved in the Ca2+-independent and PtdIns(4,5)P2-dependent binding activity of the domain. Furthermore, when this experiment was performed in the presence of 100 μm CaCl2, the PKCα-C2-K209A/K211A mutant was able to bind only 5.3 nmol of lipids at high concentrations of PtdIns(4,5)P2, which amounted to only 58% of the lipid bound with wild-type protein under these conditions (Fig. 2 D). In summary, these data support a double-site model for PtdIns(4,5)P2 binding. The first and most important would be the lysine-rich cluster, which binds PtdIns(4,5)P2 with no need for Ca2+, and the second would be located in the Ca2+ binding region. However, this last site exhibits a relatively low affinity for PtdIns(4,5)P2 even in the presence of Ca2+, suggesting that this inositide fits better in the lysine-rich region than in the Ca2+ binding region. As described in the Introduction, PKCα activation is regulated by multiple factors (10Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (851) Google Scholar). Moreover, several of the domains included in the protein are involved in the full activation of the enzyme, which further complicates the situation. To investigate the role of this new PtdIns(4,5)P2-binding site in PKCα membrane translocation, the binding of the protein to POPC multilamellar vesicles was studied by including 5 mol % PtdIns(4,5)P2 or 20 mol % POPS in the presence of Ca2+ and, in some cases, using a phorbol ester (PMA) as a C1 domain-dependent activator (Fig. 3 A). Both PtdIns(4,5)P2 and POPS were able to produce 50% protein translocation to the membranes in the absence of PMA. This translocation increased to 75% when both phospholipids were included in the same vesicles (5 mol % PIP2 and 20 mol % POPS). As expected, the inclusion of a saturating concentration of PMA (0.3 mol %) cooperated and increased the proportion of translocated protein to 100%, independently of the negatively charged phospholipids present in the membrane vesicles. Strikingly, very different results were obtained when these experiments were performed in the absence of Ca2+ (Fig. 3 B). No PKCα translocation was detected in the absence of PMA. Furthermore, when 0.3 mol % PMA was included in the assay, only 10% of protein translocation was detected in vesicles containing either POPS or PtdIns(4,5)P2/POPS, suggesting that these binding sites were not functional or accessible to the membrane vesicles under these conditions. To test this further, a PKCα substrate (histone III-S) was included in the binding reaction. In this case, protein translocation was 51, 42, and 45% when PtdIns(4,5)P2, POPS, or POPS/PtdIns(4,5)P2, respectively, were included in the vesicles, indicating that the presence of substrate enhances the ability of the PtdIns(4,5)P2 and PS sites to interact with the lipid vesicles. When PMA and histone were included in the model membranes, protein translocation to them was 100%, suggesting that under these conditions either PtdIns(4,5)P2 or POPS with PMA access their corresponding sites (C2 and C1 domain, respectively), producing full translocation of the enzyme to the membrane vesicles. To study whether protein translocation to the phospholipid vesicles correlates with its activation, PKCα specific activity was measured under conditions similar to that used in the binding assays. Fig. 4 A shows the catalytic activity of the enzyme in the presence of 100 μmCaCl2, 0.3 mol % PMA, and increasing concentrations of POPS or PtdIns(4,5)P2 in the phospholipid vesicles. Under these conditions, 20 mol % PtdIns(4,5)P2 increased enzyme activity more than 5 times that obtained when using vesicles containing 20 mol % POPS. These data correlate well with the binding assays, because 5 mol % PtdIns(4,5)P2 exhibited a similar binding and activation capacity to 20 mol % POPS. In the absence of Ca2+, vesicles containing PtdIns(4,5)P2 increased the enzyme activity more than 6 times that obtained with POPS-containing vesicles, which also suggested an important role for the PtdIns(4,5)P2-binding site in enzyme activation under these conditions (Fig. 4 B). These data correlated well with those obtained in the binding assay, because full translocation of the enzyme to the phospholipid vesicles occurred in the presence of histone and PMA. Note also that the PtdIns(4,5)P2-dependent specific activity of PKCα in the presence of Ca2+ is almost 3 times the specific activity obtained in the absence of Ca2+, indicating again that, directly or indirectly, Ca2+ plays a role in the PtdIns(4,5)P2-dependent activation of the enzyme. To address this question the same C2 domain mutants as above were used in the context of full-length PKCα, namely PKCα-D246N/D248N and PKCα-K209A/K211A to measure the effect of increasing concentrations of PtdIns(4,5)P2 and POPS on the specific enzyme activity. The results were compared with those obtained with wild-type protein. Fig. 5 A shows the activation of PKCα-D246N/D248N and PKCα-K209A/K211A in the presence of 100 μm CaCl2, 0.3 mol % PMA, and increasing concentrations of PtdIns(4,5)P2. It can be clearly observed that PKCα-K209A/K211A activation represents only 34% and PKCα-D246N/D248N 60% of the total activity exhibited by wild-type protein (Table I).Table ISpecific activity of wild-type PKCα and its mutants (nmol of Pi /min/mg)+PMA−PMA, +Ca2++Ca2+−Ca2+PKCα812275620PKCα-D246N/D248N485240276PKCα-K209A/K211A279235113Specific activity of the enzyme was measured in the presence of vesicles containing 20 mol % PtdIns(4,5)P2 and 80 mol % POPC. Histone III-S was used as a s" @default.
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• W2059335098 title "A New Phosphatidylinositol 4,5-Bisphosphate-binding Site Located in the C2 Domain of Protein Kinase Cα" @default.
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