Protein Kinase C (PKC)-induced PKC Down-regulation
1995; Elsevier BV; Volume: 270; Issue: 6 Linguagem: Inglês
10.1074/jbc.270.6.2669
ISSN1083-351X
AutoresNigel T. Goode, Nasser Hajibagheri, Peter J. Parker,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoPhorbol esters cause long term activation of protein kinase C (PKC) and frequently the down-regulation of PKC protein levels in mammalian cells. Mammalian PKC-γ, -δ, and -η down-regulate in response to phorbol esters when expressed in Schizosaccharomyces pombe. However, PKC-ε does not down-regulate in S. pombe, in contrast to the behavior of this isotype in mammalian cells. Co-expression of PKC-γ or -δ with PKC-ε in S. pombe renders PKC-ε susceptible to down-regulation. A protein kinase defective form of PKC-δ does not down-regulate efficiently in S. pombe but, like PKC-ε, is susceptible when co-expressed with PKC-γ or full-length PKC-δ. Thus, down-regulation is a consequence of the catalytic function of certain PKC isotypes with other isotypes being affected in trans. PKC down-regulation parallels a striking accumulation of vesicles in S. pombe, suggesting a direct relationship between these events. Phorbol esters cause long term activation of protein kinase C (PKC) and frequently the down-regulation of PKC protein levels in mammalian cells. Mammalian PKC-γ, -δ, and -η down-regulate in response to phorbol esters when expressed in Schizosaccharomyces pombe. However, PKC-ε does not down-regulate in S. pombe, in contrast to the behavior of this isotype in mammalian cells. Co-expression of PKC-γ or -δ with PKC-ε in S. pombe renders PKC-ε susceptible to down-regulation. A protein kinase defective form of PKC-δ does not down-regulate efficiently in S. pombe but, like PKC-ε, is susceptible when co-expressed with PKC-γ or full-length PKC-δ. Thus, down-regulation is a consequence of the catalytic function of certain PKC isotypes with other isotypes being affected in trans. PKC down-regulation parallels a striking accumulation of vesicles in S. pombe, suggesting a direct relationship between these events. INTRODUCTIONProtein kinase C (PKC) ( 1The abbreviations used are: PKCprotein kinase CDAGdiacylglycerolTPA12-O-tetradecanoylphorbol-13-acetate. )is the major cellular receptor for phorbol esters(1Ashendel C.L. Biochim. Biophys. Acta. 1985; 822: 219-242Crossref PubMed Scopus (432) Google Scholar), and most PKC isotypes can be activated by phorbol esters both in vivo and in vitro(2Stabel S. Parker P.J. Pharmacol. & Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Phorbol esters promote the binding of PKC to membranes analogous to the effect of the natural PKC activator, diacylglycerol (DAG)(3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar, 4Huang K.-P. Trends Neurol. Sci. 1989; 12: 425-431Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 5Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar, 6Zidovetzki R. Lester D.S. Biochim. Biophys. Acta. 1992; 1134: 261-272Crossref PubMed Scopus (87) Google Scholar). However, phorbol esters differ from DAG primarily by duration of action. DAG production in the membrane (for example, through activation of phospholipase C, which hydrolyzes inositol polyphosphates(7Rhee S.G. Choi K.D. J. Biol. Chem. 1992; 267: 12393-12396Abstract Full Text PDF PubMed Google Scholar)) is short-lived as the DAG is rapidly metabolized(3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Under these circumstances, PKC transiently translocates from the cytosol to the membrane, whereas phorbol esters are not metabolized and cause long term membrane association of PKC (8Nishizuka Y. Nature. 1984; 308: 693-695Crossref PubMed Scopus (5741) Google Scholar).One consequence of phorbol ester treatment, correlated with sustained association of PKC with membranes, is the down-regulation of the PKC proteins themselves(9Rodriguez-Pena A. Rozengurt E. Biochem. Biophys. Res. Commun. 1984; 120: 1053-1059Crossref PubMed Scopus (430) Google Scholar, 10Young S. Parker P.J. Ullrich A. Stabel S. Biochem. J. 1987; 244: 775-779Crossref PubMed Scopus (353) Google Scholar). Certain natural agonists can also cause the down-regulation of some PKC isotypes(11Kiley S.C. Parker P.J. Fabbro D. Jaken S. Mol. Endocrinol. 1992; 6: 120-131PubMed Google Scholar, 12Olivier A.R. Parker P.J. J. Biol. Chem. 1994; 269: 2758-2763Abstract Full Text PDF PubMed Google Scholar); down-regulation can follow physiological stimuli, possibly through the sustained production of DAG by phospholipases, including phospholipase D(12Olivier A.R. Parker P.J. J. Biol. Chem. 1994; 269: 2758-2763Abstract Full Text PDF PubMed Google Scholar, 13Kiley S.C. Parker P.J. Fabbro D. Jaken S. J. Biol. Chem. 1991; 266: 23761-23768Abstract Full Text PDF PubMed Google Scholar). However, the exact mechanism for the down-regulation of PKC has not been described. It has been proposed that Ca2+-activated neutral proteases (calpains) specifically degrade membrane-bound PKC, based on the in vitro sensitivity of PKC to various proteases(14Murray A.W. Fournier A. Hardy S.J. Trends Biochem. Sci. 1987; 12: 53-54Abstract Full Text PDF Scopus (87) Google Scholar, 15Kishimoto A. Mikawa K. Hashimoto K. Yasuda I. Tanaka S.-I. Tominaga M. Kuroda T. Nishizuka Y. J. Biol. Chem. 1989; 264: 4088-4092Abstract Full Text PDF PubMed Google Scholar).Mammalian PKC isotypes vary in their susceptibility to down-regulation when expressed in Schizosaccharomyces pombe(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). PKC-ζ protein levels were not affected by phorbol esters. PKC-ζ differs structurally from other isotypes by lacking one of the two usual cysteine repeats (17Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar) that correlate with phorbol ester binding(18Kaibuchi K. Fukumoto Y. Oku N. Takai Y. Arai K. Muramatsu M. J. Biol. Chem. 1989; 264: 13489-13496Abstract Full Text PDF PubMed Google Scholar, 19Cazaubon S. Marais R. Parker P. Strosberg A.D. Eur. J. Biochem. 1989; 182: 401-406Crossref PubMed Scopus (30) Google Scholar). This isotype does not bind, is not activated by phorbol esters in vitro(20Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar), and does not translocate to the membrane in whole cells after phorbol ester treatment(21Gschwendt M. Leibersperger H. Kittstein W. Marks F. FEBS Lett. 1992; 307: 151-155Crossref PubMed Scopus (80) Google Scholar, 22Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar, 23Kochs G. Hummel R. Meyer D. Hug H. Marme D. Sarre T.F. Eur. J. Biochem. 1993; 216: 597-606Crossref PubMed Scopus (85) Google Scholar). Thus, it is expected that PKC-ζ does not respond to phorbol esters in S. pombe. PKC-γ, -δ, and -η did down-regulate as they do in mammalian cells(3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Unexpectedly, PKC-ε did not down-regulate in S. pombe. PKC-ε binds phorbol esters (2Stabel S. Parker P.J. Pharmacol. & Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar) and does down-regulate after phorbol ester treatment in mammalian cells, although the rate of down-regulation can be slower than for other PKC isotypes(24Olivier A.R. Parker P.J. J. Cell. Physiol. 1992; 152: 240-244Crossref PubMed Scopus (77) Google Scholar). Thus, down-regulation showed a PKC isotype specificity in S. pombe that differed from that seen in mammalian cells.Here, it is shown that down-regulation requires kinase function of certain PKC isotypes, and isotypes that cannot themselves initiate the process can be affected by susceptible isotypes. Thus, the process is of a general nature but occurs in response only to certain isotypes. Furthermore, down-regulation parallels the accumulation of vesicles, suggesting an association between these events.RESULTS AND DISCUSSIONPKC-γ, -δ, and -η down-regulate in response to TPA in both S. pombe and mammalian cells(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). However, TPA does not cause the down-regulation of PKC-ε in S. pombe(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar) in contrast to the effect in mammalian cells(24Olivier A.R. Parker P.J. J. Cell. Physiol. 1992; 152: 240-244Crossref PubMed Scopus (77) Google Scholar). All four of these enzymes are phorbol ester-dependent protein kinases when purified from S. pombe(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). Thus, the ability to down-regulate does not simply reflect PKC activity or phorbol binding but is specified by the isotype. Most mammalian cells express several PKC isotypes(2Stabel S. Parker P.J. Pharmacol. & Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar), and down-regulation of PKC-ε in these cells may result from the function of other isotypes. This hypothesis was tested by expressing additional PKCs with PKC-ε in S. pombe. S. pombe was used because of its low level of endogenous TPA-dependent PKC activity(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar), which reduces the possible effects of one isotype upon another.A stable strain of S. pombe expressing PKC-ε (16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar) was supertransformed with vectors to express PKC-δ or -ζ. In vector control cultures, PKC-ε does not down-regulate in response to TPA (Fig. 1, lowerpanels, lanes1-4 and 9-12). Co-expression of PKC-ζ did not lead to PKC-ε down-regulation, and PKC-ζ itself did not down-regulate (Fig. 1, lanes13-16). In contrast, TPA treatment of cells co-expressing PKC-δ with PKC-ε led to a marked decrease in PKC-ε protein levels in parallel with the down-regulation of PKC-δ (Fig. 1, lanes5-8; Table 1). Thus, PKC down-regulation is a consequence of the action of only some PKC isotypes (δ), and other isotypes (ε) can be affected in trans.Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab It is unlikely that down-regulation simply follows relocation of the enzyme to the membrane since both PKC-δ and -ε translocate to membranes after TPA treatment in mammalian cells (2Stabel S. Parker P.J. Pharmacol. & Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar) and both show TPA-dependent kinase activity on extraction from S. pombe(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). A function of PKC-δ and not of PKC-ε must drive down-regulation. To establish whether this function involved the active kinase domain of PKC-δ, a kinase-defective form of PKC-δ (PKC-δΔ) was transformed into S. pombe. In contrast to full-length PKC-δ, the inactive form did not efficiently down-regulate in response to TPA (Fig. 2, lanes1-4). However, the kinase-defective mutant did down-regulate when co-expressed with full-length PKC-δ (Fig. 2, lanes5-8; Table 1). Thus, the response requires kinase activity, as distinct from the physical presence, of certain isotypes (e.g. δ). Other isotypes (e.g. ε) or indeed catalytically inactive mutants (e.g. δΔ) can be induced to down-regulate, but isotype-specific kinase function is required to initiate the process.Figure 2PKC-δΔ down-regulates when co-expressed with full-length PKC-δ. A cell line expressing PKC-δΔ was transformed with vector or with full-length PKC-δ. Two isolates are shown for each condition. Western blot analysis, using the PKC-δ antibody, was performed on extracts from cells cultured for 30 h in the presence or absence of TPA (±). The positions of PKC-δΔ and -δ are shown. PKC-δΔ has a molecular mass of 64 kDa. Lanes 1-4, PKC-δΔ+ vector; lanes 5-8, PKC-δΔ+ PKC-δ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to PKC-δ, the γ and η isotypes also down-regulate in S. pombe(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). To ascertain if the trans dominant effect on down-regulation was specific to the PKC-δ/-ε combination or applied more generally, the effect of PKC-γ on the down-regulation of PKC-δΔ and -ε was determined. Co-expression of PKC-γ with PKC-δΔ markedly increased the extent of down-regulation of the mutant PKC-δ protein after TPA treatment (Table 1). Similarly, down-regulation of PKC-ε was increased when co-expressed with PKC-γ (Table 1). In both instances, PKC-γ effectively down-regulated after TPA treatment (data not shown). Thus, PKC-γ, like PKC-δ, can drive the down-regulation of other isotypes (PKC-ε and -δΔ). This trans dominant effect, however, does not apply to all isotypes. PKC-ζ does not down-regulate when expressed alone or with PKC-δ in S. pombe (Fig. 3A, lanes1-4 and 5-8, respectively; Table 1). The lack of trans effect of PKC-δ on PKC-ζ also establishes that down-regulation is not due to a repressive effect of PKC-δ on the promoter used to express the PKCs or on PKC translation.Figure 3PKC down-regulation is not due to promoter or growth effects. A, PKC-δ does not cause the down-regulation of PKC-ζ. The PKC-ζ cell line was transformed with vector or PKC-δ. Two isolates are shown for each condition. Extracts were made from cells after culture for 30 h in the presence or absence of TPA (±). Positions of PKC-δ and -ζ are indicated. The two duplicate membranes were analyzed with the PKC-δ and -ζ antibodies, respectively. Lanes 1-4, PKC-ζ+ vector; lanes 5-8, PKC-ζ+ PKC-δ. B, growth arrest does not account for down-regulation. A stable PKC-δ S. pombe cell line (16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar) was cultured in nitrogen-replete (lanes 1-2) or nitrogen-deficient (lanes 3-4) medium. After 24 h, the cultures were continued for a further 30 h in the presence or absence of TPA (±). Extracts were then made and examined by Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Down-regulation correlates with a TPA-dependent growth inhibition(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). Therefore, it was important to determine that the loss of PKC protein does not simply follow growth arrest. PKC-δ expressing S. pombe were cultured in nitrogen-deficient or nitrogen-replete medium for 54 h. PKC-δ is expressed at similar levels in growth-arrested and growing cells (Fig. 3B, lanes1 and 3). Furthermore, in both instances, treatment of these arrested cells with TPA led to PKC-δ down-regulation (Fig. 3B, lanes2 and 4). Thus, down-regulation does not automatically follow growth arrest but requires TPA treatment of specific isotypes.Three distinct PKC down-regulation responses are identified. PKC-ζ is not affected, as predicted by its unresponsiveness to TPA. PKC-ε translocates to the membrane in mammalian cells but does not trigger down-regulation, at least in S. pombe. Down-regulation can be initiated by both PKC-γ and -δ, and, in addition to causing homologous down-regulation, these isotypes can affect other members of the PKC family (PKC-δΔ and -ε). Furthermore, since PKC down-regulation is initiated by active kinase, the process appears to constitute a direct negative feedback pathway through the destruction of the active PKC. However, whether the ability of PKC to drive this process is a negative or positive signal remains an open issue.Inactive PKC-α and -γ mutants can down-regulate after TPA treatment when expressed in mammalian cells(31Pears C. Parker P.J. FEBS Lett. 1991; 284: 120-122Crossref PubMed Scopus (21) Google Scholar, 32Freisewinkel I. Riethmacher D. Stabel S. FEBS Lett. 1991; 280: 262-266Crossref PubMed Scopus (26) Google Scholar). This apparent contradiction with the results shown above for PKC-δΔ can be explained by the presence of endogenous mammalian PKCs (which trigger down-regulation) in the cell types used. Interestingly, others have shown that an inactive PKC-α mutant does not down-regulate when expressed in mammalian cells(33Ohno S. Konno Y. Akita Y. Yano A. Suzuki K. J. Biol. Chem. 1990; 265: 6296-6300Abstract Full Text PDF PubMed Google Scholar). Different levels of endogenous PKCs capable of triggering and/or sustaining down-regulation in the cell lines used in these experiments could account for the conflicting results(31Pears C. Parker P.J. FEBS Lett. 1991; 284: 120-122Crossref PubMed Scopus (21) Google Scholar, 33Ohno S. Konno Y. Akita Y. Yano A. Suzuki K. J. Biol. Chem. 1990; 265: 6296-6300Abstract Full Text PDF PubMed Google Scholar). Efficient down-regulation would not occur in cell lines with low PKC activity, a situation analogous to S. pombe cells that contain low levels of endogenous PKC activity(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar).PKC isotype-specific functions are apparent since PKC-ε is clearly distinguished from both PKC-γ and -δ in its inability to down-regulate in isolation, despite these isotypes being similar in terms of TPA regulation of activity. The implication is that the output signals must differ. The specific molecular events have not been elucidated, but a correlation was noted between the ability to down-regulate and a marked vesicle accumulation in S. pombe (of which at least an element was endocytic)(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). Cells expressing PKC-δΔ, -ε, and -ζ did not accumulate vesicles even after TPA treatment(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). To determine if down-regulation of PKC-δΔ correlated with vesicle accumulation, PKC-δΔ cells supertransformed with vector, PKC-γ, or PKC-δ, were examined after culture with or without TPA. PKC-δΔ cells transfected with the control vector did not accumulate vesicles at any point. However, cells co-expressing PKC-γ accumulated vesicles in a phorbol ester-dependent fashion with 42% of cells affected 3 h after addition of TPA (Fig. 4). In cells where PKC-δ was co-expressed with PKC-δΔ, vesicles were evident in 8.5% of cells, and this percentage was increased to 31% after treatment with TPA for 3 h (Fig. 4). Electron micrographs of representative cultures are shown in Fig. 5. Thus, TPA treatment of cells co-expressing PKC-γ or -δ with PKC-δΔ led to vesicle accumulation in parallel with PKC-δ mutant down-regulation. It is hypothesized that the up-regulation of membrane transport processes, including endocytosis, is central to the down-regulation of PKC. Activated PKCs associate with membranes; increased endocytic activity would lead to an increased rate of traffic to and from the plasma membrane, and the PKCs are presumably targeted for degradation in vacuoles (or lysosomes) or perhaps exposure to proteasomes(34Gruenberg J. Clague M.J. Curr. Opin. Cell Biol. 1992; 4: 593-599Crossref PubMed Scopus (48) Google Scholar, 35Pryer N.K. Wuesthube L.J. Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (368) Google Scholar).Figure 4Expression of PKC-γ or -δ in PKC-δΔ cells induces vesicle accumulation. PKC-δΔ-expressing S. pombe cells were transformed with vector, PKC-γ, or PKC-δ. After culture for 44 h in minimal selective medium (containing TPA for the final period as indicated), the cells were fixed and examined by electron microscopy. Results are expressed as the percentage of cells with visible vesicle accumulation. Two samples of 100 random cells were examined for each condition, and results are the mean and range. At no stage were vesicles seen in the vector control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Representative cell sections of the PKC-δΔ transformants. The cells (as used for Fig. 4) are PKC-δΔ+ vector (A) and PKC-δΔ+ PKC-γ (B). Both cells were treated with TPA for 3 h. Co-expression of PKC-γ induces vesicle accumulation. CW, cell wall; G, Golgi apparatus; M, mitochondrion; N, nucleus; PM, plasma membrane; V, vacuole; VE, vesicles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This hypothesis requires membrane localization of PKC as a prerequisite for down-regulation. PKC-ζ does not down-regulate following TPA treatment, even if co-expressed with PKC-δ, possibly reflecting the fact that PKC-ζ does not translocate to the membrane in response to TPA(21Gschwendt M. Leibersperger H. Kittstein W. Marks F. FEBS Lett. 1992; 307: 151-155Crossref PubMed Scopus (80) Google Scholar, 22Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar, 23Kochs G. Hummel R. Meyer D. Hug H. Marme D. Sarre T.F. Eur. J. Biochem. 1993; 216: 597-606Crossref PubMed Scopus (85) Google Scholar). However, a chimeric molecule containing the regulatory domain of PKC-δ fused to the catalytic domain of PKC-ζ down-regulated when expressed in S. pombe(36Goode N.T. Parker P.J. FEBS Lett. 1994; 340: 145-150Crossref PubMed Scopus (16) Google Scholar). The PKC-δ regulatory domain of this chimera would specify membrane localization after TPA treatment. These data support the suggestion that PKC down-regulation is a membrane-driven process.It is noted that vesicles have been seen where down-regulation was not obvious. PKC-δ cells accumulate vesicles (Fig. 4), but down-regulation occurs only after TPA treatment. Since down-regulation by necessity reflects a balance between the rates of synthesis and destruction, the increased rate of breakdown due to increased vesicle traffic after TPA treatment (Fig. 4) would lead to a net loss of PKC-δ protein in these cells. This effect is probably accentuated because phorbol esters cause a tight association of PKC with membranes (3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar, 6Zidovetzki R. Lester D.S. Biochim. Biophys. Acta. 1992; 1134: 261-272Crossref PubMed Scopus (87) Google Scholar); the PKCs would remain attached to membranes up to and including the time of sorting to degradative compartments. The idea that PKC down-regulation is part of a nonspecific degradative process is supported by recent work showing that specific proteases cannot account for down-regulation(37Junco M. Webster C. Bosca L. Parker P.J. Eur. J. Biochem. 1994; 223: 259-263Crossref PubMed Scopus (28) Google Scholar).In summary, PKC down-regulation is a function of the kinase activity of certain isotypes. Isotypes that cannot initiate the down-regulation process can be affected in trans, predicting that some PKC isotypes contribute to the control of action of other PKCs. The strict correlation between the up-regulation of vesicle traffic and the ability to down-regulate suggests these two phenomena are related and that PKC down-regulation occurs by the same general process as PKC-mediated receptor internalization and down-regulation. INTRODUCTIONProtein kinase C (PKC) ( 1The abbreviations used are: PKCprotein kinase CDAGdiacylglycerolTPA12-O-tetradecanoylphorbol-13-acetate. )is the major cellular receptor for phorbol esters(1Ashendel C.L. Biochim. Biophys. Acta. 1985; 822: 219-242Crossref PubMed Scopus (432) Google Scholar), and most PKC isotypes can be activated by phorbol esters both in vivo and in vitro(2Stabel S. Parker P.J. Pharmacol. & Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Phorbol esters promote the binding of PKC to membranes analogous to the effect of the natural PKC activator, diacylglycerol (DAG)(3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar, 4Huang K.-P. Trends Neurol. Sci. 1989; 12: 425-431Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 5Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar, 6Zidovetzki R. Lester D.S. Biochim. Biophys. Acta. 1992; 1134: 261-272Crossref PubMed Scopus (87) Google Scholar). However, phorbol esters differ from DAG primarily by duration of action. DAG production in the membrane (for example, through activation of phospholipase C, which hydrolyzes inositol polyphosphates(7Rhee S.G. Choi K.D. J. Biol. Chem. 1992; 267: 12393-12396Abstract Full Text PDF PubMed Google Scholar)) is short-lived as the DAG is rapidly metabolized(3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Under these circumstances, PKC transiently translocates from the cytosol to the membrane, whereas phorbol esters are not metabolized and cause long term membrane association of PKC (8Nishizuka Y. Nature. 1984; 308: 693-695Crossref PubMed Scopus (5741) Google Scholar).One consequence of phorbol ester treatment, correlated with sustained association of PKC with membranes, is the down-regulation of the PKC proteins themselves(9Rodriguez-Pena A. Rozengurt E. Biochem. Biophys. Res. Commun. 1984; 120: 1053-1059Crossref PubMed Scopus (430) Google Scholar, 10Young S. Parker P.J. Ullrich A. Stabel S. Biochem. J. 1987; 244: 775-779Crossref PubMed Scopus (353) Google Scholar). Certain natural agonists can also cause the down-regulation of some PKC isotypes(11Kiley S.C. Parker P.J. Fabbro D. Jaken S. Mol. Endocrinol. 1992; 6: 120-131PubMed Google Scholar, 12Olivier A.R. Parker P.J. J. Biol. Chem. 1994; 269: 2758-2763Abstract Full Text PDF PubMed Google Scholar); down-regulation can follow physiological stimuli, possibly through the sustained production of DAG by phospholipases, including phospholipase D(12Olivier A.R. Parker P.J. J. Biol. Chem. 1994; 269: 2758-2763Abstract Full Text PDF PubMed Google Scholar, 13Kiley S.C. Parker P.J. Fabbro D. Jaken S. J. Biol. Chem. 1991; 266: 23761-23768Abstract Full Text PDF PubMed Google Scholar). However, the exact mechanism for the down-regulation of PKC has not been described. It has been proposed that Ca2+-activated neutral proteases (calpains) specifically degrade membrane-bound PKC, based on the in vitro sensitivity of PKC to various proteases(14Murray A.W. Fournier A. Hardy S.J. Trends Biochem. Sci. 1987; 12: 53-54Abstract Full Text PDF Scopus (87) Google Scholar, 15Kishimoto A. Mikawa K. Hashimoto K. Yasuda I. Tanaka S.-I. Tominaga M. Kuroda T. Nishizuka Y. J. Biol. Chem. 1989; 264: 4088-4092Abstract Full Text PDF PubMed Google Scholar).Mammalian PKC isotypes vary in their susceptibility to down-regulation when expressed in Schizosaccharomyces pombe(16Goode N.T. Hajibagheri M.A.N. Warren G. Parker P.J. Mol. Biol. Cell. 1994; 5: 907-920Crossref PubMed Scopus (29) Google Scholar). PKC-ζ protein levels were not affected by phorbol esters. PKC-ζ differs structurally from other isotypes by lacking one of the two usual cysteine repeats (17Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar) that correlate with phorbol ester binding(18Kaibuchi K. Fukumoto Y. Oku N. Takai Y. Arai K. Muramatsu M. J. Biol. Chem. 1989; 264: 13489-13496Abstract Full Text PDF PubMed Google Scholar, 19Cazaubon S. Marais R. Parker P. Strosberg A.D. Eur. J. Biochem. 1989; 182: 401-406Crossref PubMed Scopus (30) Google Scholar). This isotype does not bind, is not activated by phorbol esters in vitro(20Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar), and does not translocate to the membrane in whole cells after phorbol ester treatment(21Gschwendt M. Leibersperger H. Kittstein W. Marks F. FEBS Lett. 1992; 307: 151-155Crossref PubMed Scopus (80) Google Scholar, 22Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar, 23Kochs G. Hummel R. Meyer D. Hug H. Marme D. Sarre T.F. Eur. J. Biochem. 1993; 216: 597-606Crossref PubMed Scopus (85) Google Scholar). Thus, it is expected that PKC-ζ does not respond to phorbol esters in S. pombe. PKC-γ, -δ, and -η did down-regulate as they do in mammalian cells(3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar). Unexpectedly, PKC-ε did not down-regulate in S. pombe. PKC-ε binds phorbol esters (2Stabel S. Parker P.J. Pharmacol. & Ther. 1991; 51: 71-95Crossref PubMed Scopus (452) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4214) Google Scholar) and does down-regulate after phorbol ester treatment in mammalian cells, although the rate of down-regulation can be slower than for other PKC isotypes(24Olivier A.R. Parker P.J. J. Cell. Physiol. 1992; 152: 240-244Crossref PubMed Scopus (77) Google Scholar). Thus, down-regulation showed a PKC isotype specificity in S. pombe that differed from that seen in mammalian cells.Here, it is shown that down-regulation requires kinase function of certain PKC isotypes, and isotypes that cannot themselves initiate the process can be affected by susceptible isotypes. Thus, the process is of a general nature but occurs in response only to certain isotypes. Furthermore, down-regulation parallels the accumulation of vesicles, suggesting an association between these events.
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