Expression of Four Protein Kinase C Isoforms in Rat Fibroblasts

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L. L. Hsieh and I. B. Weinstein, personal communication. (19,20) was not ...... Housey, G. M., Johnson, M. D., Hsiao, W.-L. W., O'Brian, C. A.,. Murphy, J. P. ...
THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 267, No. 18, Issue of June 25, pp. 12892-12899,1992 Printed in U.S.A.

0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of Four Protein Kinase C Isoforms in Rat Fibroblasts DISTINCTSUBCELLULARDISTRIBUTIONANDREGULATION

BY CALCIUMANDPHORBOLESTERS* (Received for publication, June 10, 1991)

Christoph Borner$$,Sarah Nichols GuadagnoSlI, Doriano Fabbroll , and I. Bernard Weinstein$** From the $Comprehensive Cancer Center and Institute for Cancer Research, Columbia University, the IIDepartment of Pharmacology, Columbia University, New York, New York 10032, and the I(Department of Pharmaceutical Research, Oncology K125, Ciba Geigy, Ltd., CH-4002 Basel, Switzerland

Protein kinase C (PKC), the major receptor for tuProtein kinase C (PKC)’ is one of the major mediators of mor-promoting phorbol esters, consists of a family of signals generated upon external stimulation of cells by hora t least eight distinct lipid-regulated enzymes. How the mones, neurotransmitters, and growth factors (1-3). These various PKC isozymes are regulated in vivo and how signals induce the hydrolysis of various phospholipids to they couple to particular cellular responses is largely generate diacylglycerol (DAG), an endogenous activator of unknown. We have examined the expression and reg- PKC. If phosphoinositides are hydrolyzed, another product, ulation of PKC isoforms in R 6 rat embryo fibroblasts. inositol 1,4,5-trisphosphate (IP,) may be formed which leads Northern and Western blot analyses indicate that theseto an increase in intracellular Ca2+ (4). Ca2+ is thought to cells expressfourPKC isoforms,cPKCa,nPKCt, contribute to PKC activation by facilitating the interaction nPKCS, and nPKC{; of which nPKCt and nPKCG are of the enzyme with the lipid bilayer and hence with acidic the most abundant. In agreement with the simultaneous phospholipids and DAG (5). Thismembraneassociation/ presence of cPKC and nPKC isozymes, both Ca2+-deactivation event is in most cases reversible and transient due pendentand-independent PKC activitieswere deof DAG and IP3 (5,6). Phorbol esters tected in extracts of these cells. cPKCa and nPKC{ to the rapid metabolism such as 12-0-tetradecanoyl phorbol13-acetate(TPA)can were predominantly localizedin thecytosol when subcellular fractionation was carried out in the presence substitute for DAG in the activation of PKC (7, 8). Due to stability, they are able irreversibly to of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. their potency andin vivo lipid bilayerthereby causinga cumulative When cell lysis was carried out in the presence of Ca2+, insert PKC into the >SO% of cPKCa redistributed to the particulate frac- and long-term stimulation of the enzyme (5, 9). This activation, whereas nPKC{ remained in thecytosol. In con- tion is eventuallyterminated by a subsequent proteolytic trast to cPKCm and nPKC{, 60-8070 ofnPKCc and degradation (“down-regulation”)of PKC (1-3, 6). nPKCG were located ina Ca2+-insensitive, membraneMolecular cloning has revealed the presence of multiple, bound form. Treatment of R 6 cells with 12-0-tetraclosely related PKC isoforms (for reviews, see Refs. 3, 6, and decanoylphorbol13-acetate(TPA),resultedinthe 10). With one notable exception ( l l ) , these isoforms are all translocation of all four PKC isozymes to the mem- activated by phospholipids and DAG (or TPA) albeit with brane fraction, and the subsequent down-regulation of slightly different kinetics(10-18) The only marked difference cPKCa, nPKC{, and nPKCG, nPKCt, however, wasonly between them is their sensitivity toCa2+:conventional PKCs partiallydown-regulatedinresponsetolong-term (cPKCa, PI, PII, y) are dependent on Ca2+ for activity (13TPA exposure. Overproduction of exogenous cPKCBI 16), whereas the novel PKCs (nPKCc, 6, {, q/L) are not (11, in R 6 cells conferred partial resistance of nPKCG to 12, 17-22). The different tissue distributionof PKC isoforms TPA-induced down-regulation and potentiated there- (3,23) combined with the finding that more than oneisoform sistance of nPKCc to down-regulation. These results is expressed in a single cell type (2, 3, 23-39) suggest that demonstrate that the multiple isoformsof PKC which each isoform may exert a distinct cellular function.It is, coexist within a single cell typeare differentially reghowever, unknown whether specificity for such a function is ulated by extra- and intracellular stimuli and may thereby influence growth control and transformation determined by differential in vivo activation or differential linkage to substrates (12, 40, 41) and/or signalling pathways via distinct mechanisms. of each isoform. We have investigated these possibilities by studying the regulation of PKC isoforms in rat fibroblasts. These cells were chosen since they are mitogenic to various agents which activate PKC(42) and therefore PKC activation * This work was supported in part by National Cancer Institute to control.We show here that rat embryo Grant CA 02656, an award from the Markey Charitable Trust (to B. can be linked growth fibroblasts express one cPKC and three nPKC isoforms which W.), and by Swiss National Science Foundation Grant 32-26429.89 subcellular localization,and (to D. F.). The costs of publication of this article were defrayed in differ in their relative abundance, part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. 5 Recipient of a Postdoctoral Fellowship 83.625.0.88from the Swiss National Science Foundation. Current address: Institute of Biochemistry, University of Lausanne, 155Ch. des Boveresses, CH-1066 Epalinges, Switzerland. ** To whom correspondence should be addressed: Comprehensive Cancer Center and Institute for Cancer Research, Columbia University, 701 W. 168th St., New York, NY 10032. Tel.: 212-305-6924;Fax: 212-305-6889.

The abbreviations used are: PKC, protein kinase C; cPKC, conventional Ca2+-dependent proteinkinase C; nPKC, novel Ca*+-independentprotein kinase C; IPS, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PS, phosphatidylserine; TPA, 12-0-tetradecanoyl phorboll3-acetate; Ae-pep, synthetic peptide encompassing the pseudosubstrate region of nPKCe; EGTA, [ethylenebis(oxyethylenenitri1o)ltetraacetic acid SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; kb, kilobases; bp, base pair(s);ELISA, enzyme-linked immunosorbent assay; TN, Tris-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium.

12892

C Isoforms in Rat Fibroblasts

Protein Kinase

kb

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(19,20) was not addressed in this study due to thelack of the appropriate reagents. To confirm the presence of these four PKC isoforms in R6 fibroblasts we performed Western blot analysis of total protein extracts, using isoform-specific antibodies. We detected the expression of cPKCa, nPKCt, and nPKCl as proteins with apparent molecular masses of 81, 89, and 72 kDa, respectively (Fig. 2). nPKCG was recognized as a protein doublet kD

3.2 97

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FIG. 1. Expression of themRNA transcripts of the four PKC isoforms. Northernblots showing therelative levels of cPKCa, nPKC(,nPKC6,andnPKCcmRNAsinR6embryofibroblasts. Poly(A)+ RNA was prepared for the detection of n P K C c for the other isoforms total RNA wasused. The PKCisoform-specific cDNA probes were :"'P-labeled and hybridized as described under "Experimental Procedures." Numbers on the side of the autoradiographs represent the sizes of the transcripts in kilobases; 8.1 and 3.5 kb for cPKCa; 4.1 and 2.4 for n P K C c 3.2 for nPKC6; 7.5 for nPKCc. The a nPKCc-specific cDNA probe (RP16) 2.0-kb transcript detected with is unrelated to nPKCe (see text).

regulation by Ca2+-and phorbol esters. These results unveil a n unexpected PKC heterogeneity in fibroblasts and suggest that each PKC isoform may provide a differential contribution to growth control.

RESULTS

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EXPERIMENTAL PROCEDURES*

Rat Embryo Fibroblusts Express a t Least Four PKC Isoforms-Previous studies indicated that rodent fibroblasts express the cPKCa (49, 60, 61) and nPKCt isoforms (49). To examine the possibility that the rat embryo fibroblastcell line R6 expresses other PKC isoforms, we analyzed cellular extracts for the amounts of isoform-specific mRNA, protein, and protein kinase activities. For this purpose, we obtained cDNA probes for each PKC isoform and generated polyclonal antibodies against peptides encompassing the carboxyl-terminal end (V5) of each isoform. Northern blot analyses of total RNA revealed that R6 cells express two transcripts for cPKCa of3.5 and 8.1 kb, two transcripts for nPKC{ of2.4 and 4.1 kb, one transcript for nPKCG of 3.2 kb, and one transcript for nPKCt of 7.5 kb (Fig. 1).A second transcript of -2.0 kb which was detected with our nPKCt cDNA probe (RP16) is a contaminant unrelated to nPKCt (this probe is a recombinant of nPKCt and an unrelated gene): Hybridizationwitha full length mouse nPKCt cDNA detected a single transcript of 7.5 kb (data not shown). Similar transcript sizes for the individual PKC isoforms have been reported previously in other cell systems (10, 11,17,21-25,31-33,37). Transcripts for cPKCP and cPKCr were not detectedin thesecells even when poly(A)+-RNAwas analyzed (data not shown), and the expression of nPKCv/L

12893

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FIG. 2. Expression of the proteins ofthe four PKC isoforms. Protein immunoblots showing the relative levels of cPKCa, nPKC(, nPKC6, and nPKCc protein in R6 embryo fibroblasts. Total protein extracted with hot SDS was analyzed. Immunodetection with PKCspecific antibodies was as describedunder"ExperimentalProcedures." For cPKCa, two antisera were used andgave identical results: a cPKCa-specific monoclonal antibody (Seikagaku (44)) and a monoclonal antibody raised against a peptide of the V3 region of bovine cPKCa (Amersham, cione MC5 (43)). We (49) and others (33) previously reported that the latter antibody specifically recognizes the cPKCa isozyme in rodents. cPKCn (+) or pep (+) designates preabsorption of the respective antiserum with purified cPKCa or the isoform-specific peptide antigensbefore incubation with the nitrocellulose membrane, respectively. nPKCc baculo indicates that a pure preparation of nPKCc expressed in insect cells by baculovirus-me'Portions of this paper (including "Experimental Procedures," part diated gene transfer was analyzed in parallel with the total protein of "Results," Table I, and Footnote 3) are presented in miniprintat extract from R6 cells. Brain indicates a partially purified extract of the end of this paper. Miniprint is easily read with the aid of a PKC from rat brain. The apparent molecular masses in kilodaltons standard magnifying glass. Full size photocopies are included in the of the PKC isoforms are indicated p81 for cPKCa, p72 for nPKCT, p85/72for nPKCc-like Press. p74/76for nPKC6,p89fornPKCe,and microfilm edition of the Journal that is available from Waverly proteins. L. L. Hsieh and I. B. Weinstein, personal communication.

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Protein Kinase

C Isoforms in Rat Fibroblasts

of 74 and 76 kDa. Although these molecular masses were slightly lower than those previously reported for the respective brain enzymes they are inagreement with previously reported sizes from cells overexpressing each isoform individually (11, 12, 18, 25, 32, 41, 62, 63). The identities of the different PKC isoforms were further established by the following experiments: (i) Immunodetection of the protein bands of nPKC6, nPKCt, and nPKC{ was blocked in the presence of 100 pg of the respective peptide antigen (Fig. 2), and these bands did not appear when preimmune serum was used instead of antiserum (data notshown). (ii) For cPKCa, preabsorption of the two commercially available cPKCa-specific antibodies with highly purified cPKCa from the human breastcancer cell line MDA-MB-231 (59) prevented immunodetection of the 81kDa protein band (Fig. 2). (iii) The antipeptide antibody against nPKCt reacted with three proteins of apparent molecular masses of 89,85, and 72 kDa. The 89-kDa protein was detected in a preparation of baculovirus-expressed nPKCt (Fig. 2) and therefore reflects the nPKCt holoenzyme. The other two proteins appear to represent nPKCt-like polypeptides because they were not detected upon competition with the nPKCt-specific peptide antigen (Fig. 2), and they do not react with preimmune serum (data not shown). Proteins of similar molecular masses were previously detected in COS cells overexpressing nPKCt (25,63). (iv) When tested against a preparationcontaining baculovirus-expressed nPKC6 or nPKC{: the anti-nPKC6 and anti-nPKC{peptide antibodies specifically recognized a 74/76-kDa nPKC6 protein doublet and a single 72-kDa nPKC{ protein, respectively. Proteins of similar sizes were immunodetected recently in tissues and cells with anti-nPKC6- and anti-nPKCf-specific antibodies (17, 18, 39, 64). We did notice some cross-reactivities with two of the isoform-specific antibodies. The nPKC6 antibody showed slight immunoreactivity with 89-kDa nPKCt and 81-kDa cPKCa, and the nPKCrantibody also reacted weakly with the 81-kDa protein of cPKCa (Figs. 2, 3, and 4). However, the crossreactivities of the nPKC6 and nPKC{ antibodies were weak and did not in anyway alter themajor findings of our study. nPKC6 and nPKCt Are Predominantly Membrane-bound, but nPKC{ Is Cytosolic in a Ca2+-insensitive Manner in R6 Cells-In order to determine the subcellular distribution of the four PKC isoforms and their sensitivity to Ca2+-dependent membrane association, we lysed R6cells in buffers containing either EGTA or excess amounts of Ca2+(3 mM) and analyzed cytosolic and membrane fractions for theamount of the respective immunoreactive PKC isoforms. Consistent with previous reports (24,25) we found that 80% of cPKCa resided in the cytosol and 20% was membrane-bound, when extraction was performed in the presence of EGTA (Fig. 3). On the other hand, subcellular fractionation in the presence of Ca2+ revealed that 70% of cPKCa was located in the membrane fraction and only 30% in the cytosolic fraction (Fig. 3). The subcellular localization of all three nPKC isoforms (e, 6, and {) was independent of the Ca2+concentration in the fractionation buffer. This result extends previous studies with nPKCE (24, 25) and demonstrates that not only the subcellular distribution of nPKCe, but also that of nPKC6 and nPKC{ is insensitive to Ca2+.This is consistent with the fact that nPKC isoforms do not require Ca2+for activation and appear to lack a Ca2+binding domain (21,63). Suprisingly, we found that in R6 cells the nPKC isoforms differed in their constitutive, Ca2+-insensitivecellular localization. nPKC{ resided predominantly in the cytosol, similar to cPKCa,whereas 70-80% of nPKCt and nPKC6 were membrane-bound (Fig. 3). Recent D. Fabbro, personal communication.

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FIG.3. Subcellular distribution of the four PKC isoforms in response to CaZ+. Protein immunoblots showing the levels of cPKCa, nPKC{, nPKCG, and nPKCt in subcellular fractions of R6 cells. Cell extracts were partitioned into cytosol (Cy) and membrane ( M )fractionsusing either EGTA or Ca2+ containing buffers, as described under “Experimental Procedures.” Cytosol and membrane samples for both extraction conditions were analyzed on the same gel; autoradiographs are depicted separately for the sake of presentation. Immunodetection was as described in the legend to Fig. 2. studies have found nPKC6 to be localized primarily in the particulate fraction of a variety of tissues and cell lines (64). On the other hand, nPKCt has been shown to be primarily cytosolic in some cell types (24, 25, 31-34, 41, 63) and primarily membrane-bound in others (32, 35, 36). These data indicate that depending on the cell type, nPKCt (andpossibly nPKC6 and nPKC{) can be predominantly located in the membrane or the cytosolic fraction. All PKC Isoforms Redistribute to the Membrane in Response to TPA, and nPKC6 Appears to Undergo an Additional Modification-Since we found that nPKCe and nPKC6 are predominantly membrane-bound whengrown in conventional medium containing 10% serum, we wanted to examine whether the low cytosolic level of these two isoforms were translocated to the membrane fraction in response to treatment of R6 cells with TPA. Treatment of R6 cells with a low dose of TPA (10 nM) did not significantly affect the subcellular distribution of cPKCa and nPKC{ (Fig. 4A and data not shown). Incubation of these cells with 300 nM TPA, however, was accompanied by a rapid and extensive translocation of both cytosolic cPKCa andnPKC{ to themembrane fraction, within 30 min (Fig. 4, A and B). In contrast to cPKCa and nPKC{, cytosolic nPKCt (Fig. 4C) and nPKC6 (data not shown) were slightly shifted to the membrane fraction in response to 10 nM TPA. When cells were treated with 300 nM TPA, cytosolic levels of nPKCt and nPKC6 completely disappeared and the constitutive membrane abundance of the two isoforms was further increased (Fig. 4, C and D).This is most likely due to a membrane recruitment of the cytosolic 89-kDa nPKCc and 74-kDa nPKC6 proteins. It was difficult to determine if the 76-kDa

Protein KinaseC Isoforms in Rat Fibroblasts

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FIG. 4. Subcellular distribution and down-regulation ofPKC isoforms in response to TPA.Protein immunoblotsof cPKCn ( A ) , nPKC{ ( B ) ,nPKCc (C), and nPKC6 (D)showing the amountsof the respective PKC isoforms incytosol (Cy) and membrane ( M ) fractions from control R6-Cl or cPKCPI-overexpressing R6-PKC3 cells treated without (ctrl) or with 10 or 300 nM TPA for 30 min (30'1,6 h, or 24 h. Molecular masses of the PKCisoforms are as described in legend to Fig. 2. In C, p85 and p72 represent nPKCc-like proteins differentfrom the 89-kDa nPKCc holoenzyme immunodetected with thenPKCc-specific antiserum.

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Protein Kinase C Isoforms in Rat Fibroblasts

form of nPKCG was also translocated to the membrane due to its low abundance and its close apposition to the 74-kDa nPKCG species on SDS gels (Fig. 40). In addition to membrane association, we also noted that the level of the membrane-bound 74-kDa nPKCG declined concomitant with an increase in the 76-kDa PKCG, suggesting conversion of the former into the latter species in response to TPA (Fig. 40). In a preliminary study, we found that exposure of a TPAtreated cell extract to potato acid phosphatase resulted in the conversion of the 76-kDa band to the 74-kDa nPKCG band (data notshown). This resultsuggests that the76-kDa nPKCG may be a more highly phosphorylated form of the 74-kDa species. We also noted the disappearance of a membranebound 85-kDa nPKCt-like proteinin response to 300 nM TPA (Fig. 4C). Whether this protein is an underphosphorylated form of the 89-kDa nPKCt holoenzyme which is converted into the latter by TPA-stimulatedphosphorylationor whether its amount decreases due to proteolytic degradation remains to be determined. nPKCt Is More Resistant to TPA-induced Down-regulation-Next, it was of interest to gain insight into the extent and kinetics of TPA-stimulated down-regulation of nPKC isoforms, and to determine whether this differed from the classical down-regulation of cPKCs. Treatment of R6 cells with 10 nM TPA did not lead to a significant depletion of any of the four endogenous PKC isoforms (Fig. 4, A and C, and data not shown). In response to 300 nM TPA, however, cPKCa and nPKC{ underwent complete down-regulation. Under these conditions, the 74-kDa nPKCG appeared to be first fully converted into the76-kDa enzyme species, and this was followed by complete down-regulation of the latter species, within 6 h (Fig. 40). On the other hand, the 89-kDa form of nPKCc was more resistant to TPA-stimulated downregulation since 30-40% of this protein was still immunodetectable in the membrane fraction of cells treated with 300 nM TPA for 24 h (Fig. 4C). This result comes as a surprise given the fact that nPKCt was shown to be highly susceptible to proteolysis in vitro (65) and to down-regulation by hormones, ionomycin, and phorbol ester in other cell systems (24, 31, 32). Therefore, cell types may differ with respect to their ability to down-regulate specific PKC isoforms in response to various agonists. Overexpression of an Exogenous cPKCBI Does Not Affect Translocation Properties of Endogenous PKC Isoforms but Confers Resistance to Down-regulation of nPKCt and nPKC6 in Response to TPA-To search for parameters which might influence PKC down-regulation we examined the effect of overexpression of a specific exogenous PKC isoform on the subcellular distributionandTPA-stimulatedtranslocation and down-regulation of endogenous PKCs. For this studywe took advantage of a previously reported R6 cell derivative, R6-PKC3, which stably expresses high levels of an exogenously introduced cPKCpI isoform, and displays numerous abnormalities in growth control, including a partially transformed phenotype (48). In a separate study we found that the overexpressed cPKCPI isoform is localized mainly inthe cytoplasm and undergoes membrane translocation and extensive down-regulation in response to high doses of TPA (67). As shown in Fig. 4, no gross differences with regard to subcellular distribution and TPA-stimulated membrane translocation were apparent for any of the four endogenous PKC isoforms when R6-PKC3 cells were compared to R6-C1 control cells. Furthermore, the kinetics and extent of downregulation of cPKCa and nPKC{ in response to 300 nM TPA were similar in R6-PKC3 and controlcells (Fig. 4, A and B ) . On the other hand, the partial resistance of nPKCt to TPA-

induced down-regulation seen in R6-C1 cells was increased in R6-PKC3 cells (Fig.4C). Additionally, up to 40% of the membrane-bound nPKCG was protected from TPA-induced down-regulation in R6-PKC3 cells, even though this isoform was shown to be completely depleted from R6-Cl cells following 6 hof exposure to TPA (Fig. 40). These findings indicate that overexpression of a PKC isoform, such as cPKC@I,can alter thesusceptibility of other endogenously expressed PKC isoforms (nPKCc and nPKC6) to down-regulation by TPA. A Cytosolic nPKCc-like Protein Is Transiently Increased and Then Down-regulated in Response to TPA without Apparent Membrane Translocation-In the course of our studies, we discovered an nPKCt-like protein whose abundance was regulated by TPA. We consistently detected a 72-kDa protein in the cytosol of R6 cells which cross-reacted with the nPKCtspecific antibody (Fig. 4C). Interestingly, the amount of this protein transiently increased in the cytosol of R6-C1 cells in response to both low and high doses of TPA, and theprotein gradually disappeared thereafter (Fig. 4C). In untreated cPKCP1-overexpressing R6-PKC3 cells, the 72-kDa protein was expressed constitutively at about the same level as that found in TPA-treated R6-Clcells (Fig. 4C). This may be due to the higher basal level of activated PKC in the R6-PKC3 cells. No further induction of this protein occurred when R6PKC3 cells were treated with TPA, but its down-regulation was still apparent (Fig. 4C). The response of the 72-kDa nPKCt-like protein to TPA treatment differed from the 89-kDa nPKCt since the former protein did not appear to first undergo membrane translocation before down-regulation. In addition, treatment of R6-Cl cells with TPA produced a transient increase in the abundance of the 72-kDa nPKCt-like protein in the cytosol. The fact that, in response to TPA treatment, theincrease in the 72-kDa nPKCt-like protein in R6-Cl cells was greater than the parallel decrease in the cytosolic 89-kDa nPKCt protein (Fig. 4C) provides evidence that the 72-kDa protein is not a proteolytic product of the former protein. Additionally, it is unlikely that this band was generated by in vitro proteolysis during subcellular fractionation because the 72-kDa nPKCtlike protein was also detected when cells were extracted with hot SDS (65) (Fig. 2). It is equally unlikely that this 72-kDa protein is nPKCG or nPKC{, because, apart from its close similarity in molecular size, its biochemical properties differ from those of the latter two isoforms. It is possible, however, that this nPKCt-like protein represents a product from an alternatively spliced mRNA of the nPKCt gene, such as the reported nPKCt' (66). The unresponsiveness of the nPKCtlike protein in R6 cells to TPA-induced membrane translocation may be due to the lack of a phorbol ester binding site which has been reported to be spliced out of the nPKCt' protein (21, 66). DISCUSSION

In thepresent study we demonstrate that R6 embryo fibroblasts are more heterogeneous with respect to PKC isoforms than previously thought. By combining biochemical, immunochemical, and molecular biological techniques, we have found that these cells express at least four different PKC isoforms: cPKCa, a conventional Ca2+-dependent isoform, and nPKCf, nPKCG, and nPKCS; three novel Ca2+-independent isoforms. In an effort to assess the physiology of these multiple isoforms we have analyzed their relative abundance, Ca2+-sensitivesubcellular distribution, and phorbol ester-induced translocation and down-regulation. Due to differences in titer and hybridization efficiencies of the respective antibodies and cDNA probes it was difficult to

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Protein KinaseC Isoforms in Rat Fibroblasts precisely quantitate the relative levels of expression of the different isoforms at the protein or mRNA levels. However, analyses of autoradiographs from immunoblots incubated with similar dilutions of antibodies and exposed for identical time points provided evidence that nPKCG and nPKCt are the predominant PKC isoforms in these cells. These data call into question the previous assumption that cPKCa is the major PKC isoform expressed in fibroblasts (60, 61). nPKC isoforms may be selectively activated when agonists such as platelet-derived growth factor (68) or thrombin (69) induce the hydrolysis of phosphatidylcholine to generated DAG without a concomitant elevation in intracellular Ca2+(68-71). Interestingly, all four PKC isoforms expressed in R6 cells appeared to react similarly with respect to TPA-induced redistribution from the cytosol to the membrane fraction. This is in contrast to previous findings (27-30,39,72) in other cells where phorbol esters induced selective translocations of PKC isoforms. This effect of phorbol esters, however, varies with the cell type (27-30,39) and can therefore not be considered an intrinsic propertyof the respective isoform. We were nevertheless surprised to find that nPKC{ responded to TPA by membrane translocation given the fact that it does not appear to bind phorbol esters (11).Perhaps nPKC{ has still retained a low-affinity binding site for phorbol esters (in the form of one cysteine-rich domain) (11)and therefore high doses of TPA can trigger redistribution. Alternatively, nPKC{ may be recruited to the membrane fraction by an indirect mechanism which does not involve its directbinding to TPA. Another unexpected finding was that in R6 fibroblasts, nPKCc and nPKCG were highly membrane-bound even in the absence of treatment with agonists. In accordance with previous models concerning the activity of PKC, thismembranebound state could reflect constitutive activity of the two isoforms (5, 73). Recent findings, however, indicate that the localization of nPKCt and nPKCb can be cytosolic (24, 25, 31-34,41,63) or particulate (32,35-37,64) depending on the cell type. Since Ca2+ is not a cofactor for the membraneassociation of nPKCs, specific lipids (74, 75) or PKC binding proteins (76-79) might determine the extent of membraneintegration of these isoforms. These factors may not activate nPKCs but simply anchor or compartimentalize them in the lipid bilayer. Activation of nPKCs by agonists would then not just rely on membrane translocation but involve other membrane-based mechanisms. In contrast to cPKCa, the recruitment of cytosolic nPKCt and nPKCG to the membrane by phorbol esters appears to be a relatively insignificant event in R6 cells suggesting additional mechanisms of activation. These mechanisms of activation could involve phosphorylation of nPKCs. Indeed, it has been shown recently that insulin stimulation of nPKCt activity is associated with a modification characteristic of phosphorylation (34). In addition, PKC is phosphorylated i n uiuo (59,80) andautophosphorylation of the enzyme i n uitro endows it with a higher affinity for cofactors and substrates (81). Another factor which regulates the activity of PKC isoforms is their susceptibility to inactivation by proteolysis (downregulation). I n uitro, cPKCa and nPKCtdiffer in thisrespect, with the former being highly resistant (82) and the latter being highly susceptible (66) to proteolysis. In intact cells, however, the extent of down-regulation of a particular PKC isoform does not appear to be an intrinsic property of the isoform. In R6 cells, we found that TPAcaused a total loss of immunoreactive cPKCa, nPKC{, and nPKCG but only a 60% reduction in nPKCt. In addition, another factor, namely the overexpression of a particular PKC isoform (48) appeared to influence the degradation rates of other isoforms because we

found that both nPKCt and nPKCG were more resistant to down-regulation in R6 cells overexpressing an exogenous cPKCPI enzyme. Since the overexpressed cPKCPI is itself appreciably down-regulated in response to prolonged TPA treatment (67),it is possible that high levelsof a PKCenzyme serve to sequester a specific protease so that it cannot gain access to nPKCG andnPKCt. Alternatively, activation of cPKCPI in response to TPA may initiate a cascade of phosphorylation events which ultimately protect nPKCG and nPKCt from proteolysis. It will be offuture interest toanalyze the intracellular localization and activity state of proteases involved in PKC down-regulation in different cell types. Irrespective of the mechanisms involved, our findings indicate that prolonged treatment of cells with TPA can no longer be used as amethod for discriminating PKC-dependent from -independent cellular processes because, depending on the cellular system, individual PKC isoforms may not be completely depleted by this treatment.In the case of R6 cells which overexpress cPKCPI, it is possible that the persistent abundance of membrane-bound nPKCG and nPKCt contributes to the capacity of these cells to respond to repetitive administration of TPA,aproperty whichmay favor the acquisition of a highly transformed phenotype (48). In a related paper (65)we present evidence for differential alterations of the four endogenous PKC isoforms in oncogenetransformed R6 cells.These findings provide further evidence that the individual isoforms of PKC perform distinct functions with respect to gene expression and growth control. Acknowledgments-We thank J. Knopf and N. Mazurek for PKC cDNA probes, D. Schaap and P. Parker for the purified nPKCe preparation, W. Sossin for the Ae-pep, T. Meyer and R. Allemann, Ciba-Geigy Ltd., Basel, Switzerland, for the immunization, M. Ueffing for supplying the rat brain extracts, and R. S. Krauss for critical reading of the manuscript. REFERENCES 1. Woodgett, J. R., Hunter, T., and Gould, K.L. (1987) in Cell Membranes: Methods and Reviews (Elson, E., Frazier, W., and Glaser, L., eds) pp. 215-340, Plenum Publishing Corp., New York 2. Borner, C., and Fabbro, D. (1992) in Protein Kinase C: Current Concepts and Future Perspectiues (Lester, D., and Epand, R., eds) Ellis Honvood, Sussex, England, in press 3. Nishizuka, Y. (1988) Nature 3 3 4 , 661-665 4. Berridge, M. J. (1987) Annu. Reu. Biochem. 5 6 , 159-193 5. Nelsestuen, G . L., and Bazzi, M. D. (1991) J. Bioenerg. Biomembr. 23,43-61 6. Kikkawa, U., Kishimoto, A., and Nishizuka, Y. (1989) Annu. Reu. Biochem. 5 8 , 31-44 7. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 2 5 7 , 7847-7851 8. Weinstein, I. B. (1988) Cancer Res. 48,4135-4143 9. Anderson, W. B., Estival, A., Tapiovaara, H., and Gopalakrishna, R. (1985) in Advances in Cyclic Nucleotide and Protein Phosphorylation Research (Cooper, D.M. F., and Seamon, K.B., eds) Vol. 19, pp. 287-306, Raven, New York 10. Parker, P. J., Kour, G., Marais, R.M., Mitchell, F., Pears, C., Schaap, D., Stabel, S., and Webster, C. (1989) Mol. Cell. E n docrinol. 6 5 , 1-11 11. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989) Proc. Natl. Acud. Sci. U. S. A. 8 6 , 30993103 12. Schaap, D., and Parker, P. (1990) J. Biol. Chem. 265,7301-7307 13. Burns, D. J., Bloomenthal, J., Lee, M.-H., and Bell, R. M. (1990) J. Biol. Chem. 2 6 5 , 12044-12051 14. Patel, G., and Stabel, S. (1989) Cell. Signalling 1, 227-240 15. Hannun, Y.,and Bell, R.M. (1990) J. Biol. Chem. 2 6 5 , 29622972 16. Marais, R. M., and Parker, P. J. (1989) Eur. J. Biochem. 1 8 2 , 129-137 17. Mishak, H., Bodenteich, A,, Kolch, W., Goodnight, J., Hofer, F.,

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Murphy, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354 49. Borner, C., Guadagno, S. N., Hsieh, L.-L., Hsiao, W.-L. W., and Weinstein, I. B. (1990) Cell Growth & Differ. 1 , 653-660 50. Peterson, G. L. (1977) Anal. Biochem. 83,346-356 51. Bradford, M. M. (1976) Anal. Biochem. 8 2 , 248-254 52. Regazzi, R., Fabbro, D., Costa, S. D., Borner, C., and Eppenberger, U. (1986) Znt.J. Cancer 3 7 , 731-737 53. Laemmli, U. K (1970) Nature 2 2 7 , 680-685 54. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 7 6 , 4350-4353 55. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299 56. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 6 9 , 1408-1412 57. Housey, G. M., O’Brian, C. A., Johnson, M. D., Kirschmeier, P., and Weinstein, I. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 1065-1069 58. Uchida, T., and Filburn, C. R. (1984) J. Biol. Chem. 259,1231112314 59. Borner, C., Filipuzzi, I., Wartmann, M., Eppenberger, U., and Fabbro, D. (1989) J. Biol. Chem. 2 6 4 , 13902-13909 60. McCaffrey, P. G., and Rosner, M.R. (1987) Biochem. Biophys. Res. Commun. 146,140-146 61. Weyman, C. M., Taparowsky, E. J., Wolfson, M., and Ashendel, C. L. (1988) Cancer Res. 48,6535-6541 62. Eldar, H., Zisman, Y., Ullrich, A., and Livneh, E. (1990) J. Biol. Chem. 265,13290-13296 63. Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53,731-741 64. Leibersperger, H., Gschwendt, M., Gernold, M., and Marks, F. (1991) J. Biol. Chem. 266,1477&14784 65. Borner, C., Guadagno, S. N., Hsiao, W.-L. W., Fabbro, D., Barr, M., and Weinstein, I. B. (1991) J. Biol. Chem. 2 6 7 , 1290012910 66. Schaap, D., Hsuan, J., Totty, N., and Parker, P. J. (1990) Eur. J. Biochem. 191,431-435 67. Guadaeno. S. N.. Borner., C.., and Weinstein, I. B. (1992) J. Biol. hem., in pres$ 68. Larrodera, P., Cornet, M. E., Diaz-Meco, M. T., Lopez-Barahona, M., Diaz-Laviada, I., Guddal, P. H., Johansen, T., andMoscat, J . (1990) Cell 61,.1113-1120 69. Leach, K.L., Ruff, V.A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266,3215-3221 70. Pelech, S. L., and Vance, D. E. (1989) Trends Biochem. Sci. 14, 28-30 71. Exton. J. H. (1990) J. Biol. Chem. 2 6 5 . 1-4 72. Strulovici, B.; Daniel-Issakani, S., Oto,‘E., Nestor, J., Chan, H., and Tsou, A.-P. (1989) Biochemistry 2 8 , 3569-3576 73. Bazzi, M.D., and Nelsestuen, G. L. (1988) Biochemistry 2 7 , 7589-7593 74. El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 2 6 5 , 16437-16443 75. Dennis, E. A,, Rhee, S. G., Billah, M. M., and Hannun, Y.A. (1991) FASEB J. 5 , 2068-2077 76. Mochly-Rosen, D., Khaner, H., and Lopez, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,3997-4000 77. Mochly-Rosen, D., Khaner, H., Lopez, J., and Smith, B. L. (1991) J. Biol. Chem. 266, 14866-14868 78. Wolf, M., and Baggiolini, M.(1990) Biochem. J. 2 6 9 , 723-728 79. Hyatt, S. L., Klauck, T., and Jaken,S. (1990) Mol. Carcinogenesis 3,45-53 80. Fry, M. J., Gebhardt, A., Parker, P. J., and Foulkes, J. G. (1985) EMBO J . 4 , 3173-3178 81. Huang, K.-P., Chan, K.-F. J., Singh, T. J., Nakabayashi, H., and Huang, F. L. (1986) J. Biol. Chem. 2 6 1 , 12134-12140 82. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T., and Nishizuka, Y. (1989) J. Biol. Chem. 264,4088-4092

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SUPPLEMENTALMATERIAL T O EXPRESSlONOF FOUR PROTEINKINASE C lSOFORMS IN RAT FIBROBUSTS I.DISTINCT SUBCELLULR DISTRIBUTION AND REGULATIONBY CALCIUM AND WORBOL ESTERS Christoph &mer. Sarah Nichols Guadagno,Doriano Fabbro. and I. Bernard Weinstein washed a1 68 "C in a lo high stringency ( 0 . 1 SSC) ~ and exposed to Kodak X-AR5 tilm at -70% with intensifying screens. The cDNA probes were as follows: a, a 3.0 kb Pst i-Pst i lragment emompassing nucleotdes 314-3382 at murine cPKCa (45); e. a 2.0 kb EmRi-EcoRI tragment (RP16) (57) encornpassing nucleotides 1373.2092 01 rat nPKCo (RP16); 6,a 178 bp EmRI-EcoRI fragment encompassing nucieotides 1219-1389 of rat nPKC6 (21); {, a 185 b~ EcoRI-EcoRI fragment encompassing nucleotides 270-446 of rat nPKC< (21). The relative abundance of RNA per lane was judged to be similar by comparing the ethidium bmmide staining 01 the ribosomal bands. For further mnfirmalion, the blots were rehybridized with a probe tor an endcgenous housekeepinggene, glyceraldehyde phosphate dehydmgenase (GAPDH). In ail cases. the ethidium bmmide slaining reflectedthe resuns OMained with the GAPDH pmbe. PKC was partially purilied basically as described (47). Briefly. cytosolic or NP-40 extracted membrane tractions were applied to DEAE-Sephacei columns (1 mi) equilibrated in butler A (20mM Tns-HCI, pH 7.5, 1 mM EDTA, 1 mM EGTA, 25 vglml apmtinin, 25 wlml leupeptin. 5 pM pepstatin. 1 mM phenylmethylsulfonyltluoride). The bound enzyme was eluted With buner A containing 0.25 M NaCI. Eluates were immediately assayed lor PKC activity. For immunocampetifion assays. cytosolic PKC was highly purified lrom MDA-MB-231 human breast cancer cells by DEAE-Sephacal and PS-affinity chromatography (58) as described (47). The purified PKC preparationwas lound to mainly consist of the 81 -kD cPKCa isotorm (59). PKC activity was measured using as Subsfrates either histone Ill-S or &-pep, a substrate peptide (LNRRRGSMRRRVHOVNGH) modeled afler the phylogenetically consewed pseudosubstrate region of Aplysv nPKCc. with a serine for alanine substitut~on3.The complete react#onmixture (250 PI) contained 20 mM Tris-HCi. pH 7.5. 0.5 mM dithiothreiml, 10 mM Mg(N03)~.100 pg phosphatidylserine (PS), 10 pg 1.2diolein (DAG), 24 pM [ Y ~ ~ P I A T(0.5 P vCi), 400 pM CaC12.100 EGTA. 5 2 0 wg 01 the partially purified enzyme and 50 w histone Ill-S or 1 pM Ae-pep. Caz+-independent PKC activity was assayed in the presence 01 PS and DAG, but CaC12 was replaced by 5 mM EGTA. Basal. unstimulated PKC activity was measured in the presence of EGTA. but in the absence of activators (PS, DAG, Ca2+). FolloMng incubation lor 20 min at 32'C. a 100 vi aliquot was removed lmm the reaction mixture and applied to P-81 paper (Whatman). Papen were washed three times in 75 mM phosphoric acid to remove unincoprated ATP and dried. Bound

mMonoclonal anti-bovine

PKC antibodies (MC5) (43) and125l-labeled sheep anti-mouse and donkey anti-rabbit secondary antibodies were obtained from Amersham Corp. MDnoclonai anti-cPKCa antibodies (44) were purchased from Seikagaku America Inc. (Rockville. MD). Purified b a ~ u l o - v iexpressed ~~ nPKCe was a generous gill of Dn. Schaap and Paher (12). PKC i s o q m cDNA probes for nPKC6 and nPKCr. in vector pGEM 4 (Pmmega. Madison, WI) were a genemus gin Imm Dr. Knopf at Genetics Institute Inc. (Boston. MA). The probe tor cPKCa was obmined from a murine cPKCacDNA kindly provided byN. MBmrek (45). The pseudosubstrate peptide &-pep was synthesized as described and kindly pr0"ded by W. Sossin (Howard Hudses M e d i i InstiMe. Columbia University. New York). DEAE-SephaceIwas obtained from Pharmaaa. genetian (G418) was purchased tmm Gib-BRL. 12-Otetracedanoyl phofbol 13-aceIate (TPA) was lmm LC SeNicea Corporalon (Woburn. MA). All other chemicals were reagem grade.

--

ErarullPnc o- i0 P for nPI(Z;r. nPKC6 PL unique sequences encompassing the carboxy -terminal variable V5 rwion 01 nPKCo. nPKC6 or nPKC< were Synthesized on an Applied Biosystems 430A peptide synthesizer as tollows: nPKCr (NH2-NOEEFKGFSYFGEDLMP-COOH), nPKC6 (NH2-KGFSFVNPKYEOFLECOOH).nPKCl (NH2-GFEYINPLLLSAEESV-COOH). The peptides were coupled to Keyhole limpet hemocyanin (KLH) by piutaraldehyde as described (46) and used to immunize raWns as described (47). liten 01 peplides and cross-reactivityto peptides were tested by enzyme-linked immunosorbent assay (ELISA) using peptides coupled by glutaraldehyde to bovme 88Nm albumin (BSA). The construction 01 the CPKCPI overexpressing R6 cell line (R6-PKC3) and its vector control counterpan (R6-Ct) have been described elsewhere (48). We consistently used R6-Ct cells as the normal rat embryo libroblasts tor our experiments. Both call lines were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% can 58111111 (HyCione. Logan. UT) and 50 pglml G418 in a 37-C. 6% COP humidified incubator (48). The medium was changed ever 3 days: starving and growih to postcontluence were strictly avoided. The cells were harvested for RNA or protein when subconfluent. Subconfluent R6C1 or R6-PKC3 cells were treated with 10 nM or 300 nM TPA lor 30 min. 6 h or 24 h. Aner each incubation period, cells were extracted for cytosolic and membrane pmtein. TPA was added Io the medium in dimefhylsuitoxide(Me,SO) to gwe a final concentration of 0.1% Me2S0. Prot.ln- For protein and enzyme assays, the cells were washed three times m phosphate-butferedsaline. and either immedoately iysed in preheated (95'C) extraction buner A (20 mM Tris-HCI. pH 7.5, 2 mM EDTA, 2 mM EGTA, 6 mM B-mercaptoethanol. 25 vg/ml aprotlnln. 25 w m l leupeptin. 5 pM pepslann, 1 mM phenylmethylsultonylHuoride)containmg 1 % SDS followed by sonicalion (Iotai extracls) or scraped into icecold tuner A. lysed by Sonication and centriluged at IW,OW x g for 1 h. yieldinp the soluble cytosol and the membrane pellet (49). The membrane pellet was solubilized in preheated bulfer A containing 1 % SDS. Cytosolic protein from rat brain was prepared as described (47). The protein content in these extracts was determined according to a modilied Lomy a m y (50). For lysis in Ca2'. 3 mM CaC12 was added to the cell suspension immediaeiy prior lo wnication. as described (24). Cytosoi and membrane fractions were prepared M describsd with the exception that 3 mM EGTA was added back to the cytosol. For PKC activq assays the membrane pellet was soiubilized in tuner A containing 1% ~ce-coldNonidet P-40 (NP-40) instead 01 Preheated SDS loiiowed by sonication and re-centrifugation at 100.000 x g tor 1 h. yielding the Soluble NP-40 membrane extract and the NP-40 resistant pellet. The pellet was discarded. me pmein content in the NP-40 membrane extract was determined according to the method of Bradford (51).Membrane as well as cytosol tractions w ~ r emutineiy Supplemented with glycaml to pive a tinal concentrallon of 15% (wh') in Order to prevent loss of PKC activily (52). Aner SDS-polyacrylamide gel electrophoresis (53) on a 8% acrylahide gel. proteins were elenrobbned onto a nitrocellulose membrane (Millipore) tor 3 h at 0.4 A Current in 25 mM Tns-HCI. pH 8.3. 192 mM glycine and 10% methanol. as described by Towbin et al. (54).The membrane was subsequently stalned with 0.1% amido black to assure equal protein loading and transter. Preslained molecular weight markers (Gibco-BRL) were electrophoresed in perallel. Incubation 01 the membrane with PKC antibodies was performed essentially as described (47). Briefly. non-specitic sites were blocked in 3% BSA (traction V, Sigma), 0.2% TWO" X-100 (TX) in Tris-bunered saline (TN) (biockmg buner) tor 15 mln a1 room temperature or overnight at 4'C. Membranes Were then incubated for 90 min in the prlmary antibody. All antibodies were used at 1:530 dilutions in blocking huller. Depending on the origin of the pnmary antibody. 1251-labeled sheep-anti mouse or 125l-iabeled donkey anti-rabbit Secondary antibodies were used at a tmal dilution 01 0.3pCilmi in blocklng buner for 90 mln. In between each step of the ~mmunodetection.the membranes were washed live limes for 5 mln in TN-TX. Following the last wash, immunoblots were air-dried and autoradiographed overnighl with intenslfyng screens. Specificity of the anti-PKC antibodies was determined by preabsorking the antibodies with 5 pg 01 pure CPKCm or 01 1W pg 01 the respective peptfde antigens m 20 mM Tris-HCI. pH 7.4.0.596 deoxycholate, 0.5% Triton X-1W, 0.1% SDS. 2.5 mglml ovalbumin. 5 mM EGTA. 1 mM EDTA, 200 pM leupephn. 6W nM apmtinin, lor 2 h betore incubatingthem With the membrane (47). p RNAisolation lrom the various cell Ihes was always peIfOrmedin parallel with the extraction 01 proteins. The cells were plated at a density of 1 x 106 cells per 15 cm piale. refed the lollowlng day and collected another 48 h later. Isoiation, electrophoresls. and bloning 01 total and Poly(A)+-RNA was perlormed e x a d y as described in (55.56). The RNA blots were UV crosslinked lor 2 min and then prehybridized lor 2 hat 42 "C in 50% tormamide. 5x Denhardt's. 5x SSC. 10 mM NaP04. 0.1 K SDS. and 250 pgiml sheared salmon sperm DNA. Hybridizationwas caRied out lor 24 h in 53% lormamide. 1 x Denhard's, 5x SSC. 10 mM NaP04. 10% dextran sullate and 100pgIml sheared Salmon sperm DNA.=DNA probes tor PKC isoforms were either labeled with la-32PJdNTPs (800 Cilmmoi. Amersham) by the nick translation method (for nPKCr) or wilh [a-32PldCTP (3000Cilmmol. Amersham) by random oligonucleotide primer extension (for cPKCa. nPKC6and nPKC