Regulation of Phospholipase D in L6 Skeletal Muscle Myoblasts
1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês
10.1074/jbc.272.16.10910
ISSN1083-351X
AutoresMichael G. Thompson, Steven C. Mackie, Amanda Thom, Robert M. Palmer,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe addition of vasopressin or 12-O-tetradecanoylphorbol-13-acetate (TPA) to prelabeled L6 myoblasts elicited increases in [14C]ethanolamine release, suggesting the activation of phospholipase D activity or activities. While the effects of both agonists on intracellular release were rapid and transient, when extracellular release of [14C]ethanolamine was measured, the effect of vasopressin was again rapid and transient, whereas that of TPA was delayed but sustained. Effects of both agonists on intra- and extracellular release were inhibited by the protein kinase C (PKC) inhibitor, Ro-31-8220, and PKC down-regulation by preincubation with TPA. The formation of phosphatidylbutanol elicited by vasopressin and TPA mirrored their effects on extracellular [14C]ethanolamine release in that the former was transient, whereas the latter was sustained. Responses to both agonists were abolished by PKC down-regulation. When protein synthesis was examined, the stimulation of translation by TPA and transcription by vasopressin were inhibited by Ro-31-8220. In contrast, down-regulation of PKC inhibited the synthesis response to TPA but not vasopressin. Furthermore, following down-regulation, the effect of vasopressin was still blocked by the PKC inhibitors, Ro-31-8220 and bisindolylmaleimide. Analysis of PKC isoforms in L6 cells showed the presence of α, ε, δ, μ, ι, and ζ. Down-regulation removed both cytosolic (α) and membrane-bound (ε and δ) isoforms. Thus, the elevation of phospholipase D activity or activities induced by both TPA and vasopressin and the stimulation of translation by TPA involves PKC-α, -ε, and/or -δ. In contrast, the increase in transcription elicited by vasopressin involves μ, ι, and/or ζ. Hence, although phospholipase D may be linked to increases in translation elicited by TPA, it is not involved in the stimulation of transcription by vasopressin. The addition of vasopressin or 12-O-tetradecanoylphorbol-13-acetate (TPA) to prelabeled L6 myoblasts elicited increases in [14C]ethanolamine release, suggesting the activation of phospholipase D activity or activities. While the effects of both agonists on intracellular release were rapid and transient, when extracellular release of [14C]ethanolamine was measured, the effect of vasopressin was again rapid and transient, whereas that of TPA was delayed but sustained. Effects of both agonists on intra- and extracellular release were inhibited by the protein kinase C (PKC) inhibitor, Ro-31-8220, and PKC down-regulation by preincubation with TPA. The formation of phosphatidylbutanol elicited by vasopressin and TPA mirrored their effects on extracellular [14C]ethanolamine release in that the former was transient, whereas the latter was sustained. Responses to both agonists were abolished by PKC down-regulation. When protein synthesis was examined, the stimulation of translation by TPA and transcription by vasopressin were inhibited by Ro-31-8220. In contrast, down-regulation of PKC inhibited the synthesis response to TPA but not vasopressin. Furthermore, following down-regulation, the effect of vasopressin was still blocked by the PKC inhibitors, Ro-31-8220 and bisindolylmaleimide. Analysis of PKC isoforms in L6 cells showed the presence of α, ε, δ, μ, ι, and ζ. Down-regulation removed both cytosolic (α) and membrane-bound (ε and δ) isoforms. Thus, the elevation of phospholipase D activity or activities induced by both TPA and vasopressin and the stimulation of translation by TPA involves PKC-α, -ε, and/or -δ. In contrast, the increase in transcription elicited by vasopressin involves μ, ι, and/or ζ. Hence, although phospholipase D may be linked to increases in translation elicited by TPA, it is not involved in the stimulation of transcription by vasopressin. Loss of skeletal muscle is an acute metabolic response to infection and neoplastic disease and results from a decrease in the rate of protein synthesis and an increase in the rate of protein degradation (e.g. see Refs. 1Hasselgren P.O. Talamini M. James H. Fischer J.E. Arch. Surg. 1986; 121: 919-923Crossref Scopus (88) Google Scholar, 2Strelkov A.B.S. Fields A.L.A. Baracos V.E. Am. J. Physiol. 1989; 257: C261-C269Crossref PubMed Google Scholar, 3Beck S.A. Smith K.L. Tisdale M.J. Cancer Res. 1991; 51: 6089-6093PubMed Google Scholar). To reverse this process, an understanding of the signaling pathways regulating protein turnover is essential.We have used 12-O-tetradecanoylphorbol-13-acetate (TPA) 1The abbreviations used are: TPA, 12-O-tetradecanoylphorbol 13-acetate; PLD, phospholipase D; PA, phosphatidic acid; PtdBuOH, phosphatidylbutanol; PE, phosphatidylethanolamine; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; bIM, bisindolylmaleimide. 1The abbreviations used are: TPA, 12-O-tetradecanoylphorbol 13-acetate; PLD, phospholipase D; PA, phosphatidic acid; PtdBuOH, phosphatidylbutanol; PE, phosphatidylethanolamine; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; bIM, bisindolylmaleimide. and vasopressin to investigate the coordinate regulation of protein turnover. Previous studies in this (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar, 5Morrison K.S. Mackie S.C. Palmer R.M. Thompson M.G. J. Cell. Physiol. 1995; 165: 273-283Crossref PubMed Scopus (12) Google Scholar, 6Thompson M.G. Palmer R.M. Thom A. Garden K. Lobley G.E. Calder G. Am. J. Physiol. 1996; 270: C1875-C1879Crossref PubMed Google Scholar) and other (7Goodman M.N. Biochem. J. 1987; 247: 151-156Crossref PubMed Scopus (16) Google Scholar) laboratories have shown that these two agents both stimulate protein synthesis and reduce the release ofN τ-methylhistidine, a marker of myofibrillar protein degradation from intact skeletal muscle in vitro and skeletal muscle cells in culture. However, the mechanism(s) mediating these effects are poorly understood.Many extracellular signals such as hormones, neurotransmitters, and growth factors elicit their response by activating an intracellular signaling cascade that is initiated by the hydrolysis of membrane phospholipids (8Axelrod J. Biochem. Soc. Trans. 1990; 18: 503-507Crossref PubMed Scopus (211) Google Scholar, 9Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 10Billah M.M. Curr. Opin. Immunol. 1993; 5: 114-123Crossref PubMed Scopus (151) Google Scholar, 11Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2355) Google Scholar). In L6 skeletal muscle cells, we have demonstrated that TPA and vasopressin stimulate both protein synthesis and a phospholipase D (PLD) that degrades phosphatidylcholine. Furthermore, incubation of L6 cells with exogenous PLD mimicked the effects of TPA and vasopressin on protein synthesis, implying a link between the two events (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar). Interestingly, however, TPA increased protein synthesis only during short term (90-min) incubations, which were neither blocked by the transcription inhibitor, actinomycin D, nor accompanied by increases in RNA, implying an effect only on translation. In contrast, the synthesis response to vasopressin was detected solely over longer term (6-h) incubations, where increases in RNA content and sensitivity to actinomycin D were observed, demonstrating effects on transcription (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar).One explanation of why TPA and vasopressin produced such temporally different effects on protein synthesis involves the hydrolysis by PLD of phospholipids other than phosphatidylcholine with the production of different phosphatidic acid (PA) species that either directly or indirectly manipulate translation and transcription. Although most studies of PLD to date have focused on phosphatidylcholine, it is now clear that phosphatidylethanolamine (PE) is also a substrate for PLD. TPA has been shown to activate a PLD that degrades PE in several cell types (e.g. 12–15), while as far as we are aware, there is no published evidence demonstrating that vasopressin elicits PE hydrolysis. Thus, to gain a greater insight into the mechanism(s) through which these two agonists may elicit temporally different increases in protein synthesis, we have initially focused in this study on PE metabolism and considered the possibility that TPA and vasopressin stimulate a PLD that degrades PE in L6 skeletal muscle myoblasts.Alternative explanations of the temporally different effects of TPA, vasopressin, and exogenous PLD include the possibility that either the PLD preparation may have elicited its responses through a different mechanism, e.g. through the production of lysophosphatidic acid (16Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Abstract Full Text PDF PubMed Scopus (108) Google Scholar), or that the stimulation of PLD by TPA and/or vasopressin is not related to their effects on protein synthesis. In many cell types, studies using protein kinase C (PKC) inhibitors and down-regulation protocols have implicated PKC in the stimulation of PLD by TPA and vasopressin (e.g. see Refs. 10Billah M.M. Curr. Opin. Immunol. 1993; 5: 114-123Crossref PubMed Scopus (151) Google Scholar and 17Exton J.H. J. Biol. Chem. 1990; 265: 1-4Abstract Full Text PDF PubMed Google Scholar). In the second aspect of this study, we have used similar approaches to investigate both the regulation of PE hydrolysis by PLD and the possibility that activation of PLD can be dissociated from the stimulation of protein synthesis.DISCUSSIONWe demonstrate in this study that TPA and vasopressin stimulate [14C]ethanolamine release from prelabeled L6 myoblasts. These effects were elicited in the presence of a large excess of unlabeled ethanolamine and phosphoethanolamine, thus precluding the possibility that [14C]ethanolamine was derived from [14C]phosphoethanolamine. Furthermore, the excess unlabeled ethanolamine also explains the steady increase in extracellular [14C]ethanolamine with time in unstimulated cells. The vast majority of any ethanolamine taken up by the cells is likely to be unlabeled, and thus [14C]ethanolamine released by continuous basal PLD activity (as demonstrated by basal [3H]PtdBuOH production at the end of a 2-h incubation) is likely to accumulate.When taken together with effects on [3H]PtdBuOH formation, the data imply that both TPA and vasopressin activate a PLD that degrades PE. Furthermore, the time- and concentration-dependent release of extracellular [14C]ethanolamine in response to both agonists mirrored their effects on extracellular [3H]choline release in these cells (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar). Comparison of the time-dependent effects on [3H]PtdBuOH formation by TPA using the two different protocols suggests that some of the data in the literature and the conclusions drawn from it should be treated with caution. When sufficient butanol is present for transphosphatidylation to take place, the TPA response clearly correlates with the effect on extracellular [14C]ethanolamine release; i.e. both events are sustained for at least 2 h. Interestingly, using the modified protocol to assess [3H]PtdBuOH formation, we have found in C2C12 myoblasts that the activation of PLD by TPA continues for at least 6 h. 2K. S. Morrison and M. G. Thompson, unpublished observation. TPA also stimulates ethanolamine release within 2 min in HeLa cells (12Hii C.S.T. Edwards Y.S. Murray A.W. J. Biol. Chem. 1991; 266: 20238-20243Abstract Full Text PDF PubMed Google Scholar) and after a lag period (10 min) in HL-60 cells, NIH 3T3 fibroblasts, and baby hamster kidney cells (13Kiss Z. Anderson W.B. J. Biol. Chem. 1989; 264: 1483-1487Abstract Full Text PDF PubMed Google Scholar). In contrast, as far as we are aware, this is the first demonstration that vasopressin stimulates a PLD that degrades PE in any cell type or tissue. Many of these previous studies have measured either total ethanolamine metabolites and failed to distinguish between intra- and extracellular release (e.g. see Refs. 13Kiss Z. Anderson W.B. J. Biol. Chem. 1989; 264: 1483-1487Abstract Full Text PDF PubMed Google Scholar, 14Kiss Z. Chattopadhyay J. Pettit G.R. Biochem. J. 1991; 273: 189-194Crossref PubMed Scopus (20) Google Scholar, 28Kiss Z. Anderson W.B. J. Biol. Chem. 1990; 265: 7345-7350Abstract Full Text PDF PubMed Google Scholar, and 29Kiss Z. Biochem. J. 1992; 285: 229-233Crossref PubMed Scopus (39) Google Scholar) or have measured only extracellular release (12Hii C.S.T. Edwards Y.S. Murray A.W. J. Biol. Chem. 1991; 266: 20238-20243Abstract Full Text PDF PubMed Google Scholar). When intra- and extracellular release of choline has been measured, both vasopressin and TPA rapidly stimulated intracellular release. The vasopressin response had returned to control values within 10 min, and that for TPA was almost restored within 20 min (e.g. see Ref. 30Plevin R. Wakelam M.J.O. Biochem. J. 1992; 285: 759-766Crossref PubMed Scopus (18) Google Scholar). The different time course of intra- and extracellular release observed with TPA may reflect hydrolysis of PE pools in the inner and outer leaflet of the plasma membrane or in intracellular membranes. It might also involve the activity of both cytosolic and membrane-bound PLD activities (e.g. see Ref. 31Wang P. Anthes J.C. Siegel M.I. Egan R.W. Billah M.M. J. Biol. Chem. 1991; 266: 14877-14880Abstract Full Text PDF PubMed Google Scholar and see below). In addition to TPA and vasopressin, PE hydrolysis by PLD has also been reported to be stimulated by adenine nucleotides (28Kiss Z. Anderson W.B. J. Biol. Chem. 1990; 265: 7345-7350Abstract Full Text PDF PubMed Google Scholar), platelet-derived growth factor and bombesin (29Kiss Z. Biochem. J. 1992; 285: 229-233Crossref PubMed Scopus (39) Google Scholar), lipid A (32Harris W.E. Bursten S.L. Biochem. J. 1992; 281: 675-682Crossref PubMed Scopus (14) Google Scholar), and endothelin (33Kester M. Simonson M.S. McDermott R.G. Baldi E. Dunn M.J. J. Cell. Physiol. 1992; 150: 578-585Crossref PubMed Scopus (39) Google Scholar).Vasopressin, which generates diacylglycerol and thus stimulates PKC through inositol lipid hydrolysis (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar), and TPA, activates PLD in many cell types, suggesting a role for PKC in the activation of PLD. In this and several other studies, inhibitors of PKC and down-regulation of PKC have been shown to attenuate TPA and vasopressin-stimulated PLD activity (e.g. see Refs. 34Cabot M.C. Welsh C.I. Zhang Z. Cao Y.Z. FEBS Lett. 1989; 245: 85-90Crossref PubMed Scopus (44) Google Scholar, 35Liscovitch M. J. Biol. Chem. 1989; 264: 1450-1456Abstract Full Text PDF PubMed Google Scholar, 36Plevin R. Stewart A. Paul A. Wakelam M.J.O. Br. J. Pharmacol. 1991; 104: 39Crossref PubMed Scopus (44) Google Scholar). Our observation that down-regulation completely prevented TPA- and vasopressin-induced [3H]choline or [14C]ethanolamine release or [3H]PtdBuOH formation clearly implicates PKC in this event. This suggests that the inability of Ro-31-8220 completely to inhibit TPA-induced extracellular release and vasopressin-induced intracellular release of [14C]ethanolamine may be due to its action as a competitive inhibitor (18Davis P.D. Hill C.H. Keech E. Lawton G. Nixon J.S. Sedgwick A.D. Wadsworth J. Westmacott D. Wilkinson S.E. FEBS Lett. 1989; 259: 61-63Crossref PubMed Scopus (438) Google Scholar). Contrastingly, in other cell types, the activation of PLD by TPA may not involve PKC. For example, PKC inhibitors failed to block the activation of PLD by TPA in lymphocytes (37Cao Y.Z. Reddy C. Mastro A. Biochem. Biophys. Res. Commun. 1990; 171: 955-962Crossref PubMed Scopus (42) Google Scholar) and mast cells (38Yamada K. Kanaho Y. Kiura K. Nozawa Y. Biochem. Biophys. Res. Commun. 1991; 175: 159-164Crossref PubMed Scopus (17) Google Scholar).To gain further insight into the mechanism through which PKC regulates PLD in L6 cells, we examined which isoforms are present and subject to down-regulation. As far as we are aware, this is the first attempt to study PKC isoforms in L6 cells. A previous study in skeletal muscle and L8 skeletal muscle cells demonstrated the presence of PKC-α, -δ, and -ζ, but not PKC-β αnd -γ. Furthermore, α and δ, but not ζ were down-regulated by TPA in L8 cells (39Hong D. Huan J. Ou B. Yeh J. Saido T.C. Cheeke P.R. Forsberg N.E. Biochim. Biophys. Acta. 1995; 1267: 45-54Crossref PubMed Scopus (63) Google Scholar). These are very similar to our findings reported in this study and in C2C12 skeletal muscle cells. 3M. G. Thompson and A. Thom, unpublished observation. Other work has also reported the presence of PKC-α (40Osada S.I. Mizuno K. Saido T.C. Suzuki K. Kuroki T. Ohno S. Mol. Cell. Biol. 1992; 12: 3930-3938Crossref PubMed Google Scholar, 41Yamada K. Avignon A. Standaert M.L. Cooper D.R. Spencer B. Farese R.V. Biochem. J. 1995; 308: 177-180Crossref PubMed Scopus (63) Google Scholar), -δ (41Yamada K. Avignon A. Standaert M.L. Cooper D.R. Spencer B. Farese R.V. Biochem. J. 1995; 308: 177-180Crossref PubMed Scopus (63) Google Scholar, 42Mizuno K. Kubo K. Saido T.C. Akita Y. Osada S. Kuroki T. Ohno S. Suzuki K. Eur. J. Biochem. 1991; 202: 931-940Crossref PubMed Scopus (95) Google Scholar), and -ε (41Yamada K. Avignon A. Standaert M.L. Cooper D.R. Spencer B. Farese R.V. Biochem. J. 1995; 308: 177-180Crossref PubMed Scopus (63) Google Scholar) in skeletal muscle.The data showing a correlation between the disappearance of PKC-α, -δ, and -ε and the loss of PLD activation by TPA and vasopressin clearly implicate one or more of these isoforms in this response. Their down-regulation upon TPA treatment has been observed in other cell lines (e.g. see Refs. 39Hong D. Huan J. Ou B. Yeh J. Saido T.C. Cheeke P.R. Forsberg N.E. Biochim. Biophys. Acta. 1995; 1267: 45-54Crossref PubMed Scopus (63) Google Scholar, 43Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar, and 44Akita Y. Ohno S. Yajima Y. Suzuki K. Biochem. Biophys. Res. Commun. 1990; 172: 184-189Crossref PubMed Scopus (52) Google Scholar). Furthermore, PKC-α has been shown to activate PLD in Madin Darby canine kidney cells (45Balboa M.A. Firestein B.L. Godson C. Bell K.S. Insel P.A. J. Biol. Chem. 1994; 269: 10511-10516Abstract Full Text PDF PubMed Google Scholar) and CCL39 fibroblasts (46Conricode K.M. Smith J.L. Burns D.J. Exton J.H. FEBS Lett. 1994; 342: 149-153Crossref PubMed Scopus (77) Google Scholar), while PKC-ε has been suggested to regulate PLD activity in rat mesangial cells (47Pfeilschifter J. Huwiler A. FEBS Lett. 1993; 331: 267-271Crossref PubMed Scopus (28) Google Scholar). There is no evidence to date of a role for PKC-δ in the regulation of PLD activity. It is not yet clear from our data if TPA and vasopressin activate the same PKC and/or PLD. Interestingly, although PKC-α is largely cytosolic in L6 and other cell lines (e.g. see Ref. 43Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar), we have found PKC-ε to be membrane-associated, and this is also the case to some degree in U937 cells (48Ways D.K. Messner B.R. Garris T.O. Quin W. Cook P.P. Parker P.J. Cancer Res. 1992; 52: 5604-5609PubMed Google Scholar), rat6 fibroblasts (49Borner C. Guadagno S.N. Fabbro D. Weinstein I.B. J. Biol. Chem. 1992; 267: 12892-12899Abstract Full Text PDF PubMed Google Scholar), and renal mesangial cells (50Huwiler A. Fabbro D. Stabel S. Pfeilschifter J. FEBS Lett. 1992; 300: 259-262Crossref PubMed Scopus (59) Google Scholar). It is intriguing to suggest that isoform and/or location-specific PKC/PLD activities may be responsible for both the rapid but transient responses and the delayed but sustained stimulation observed with TPA. Such a possibility requires further investigation.We have previously shown in L6 myoblasts that the EC50 for vasopressin stimulation of transcription is 10-fold higher than that for [3H]PtdBuOH formation and [3H]choline release (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar). Data from the current study show that this is also true for [14C]ethanolamine release. Furthermore, the maximal stimulation of protein synthesis observed with exogenous PLD at 6 h was 12%, whereas an increase of 30% or more was elicited by vasopressin (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar). Thus, while data in this and our previous study (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar) support a link between the activation of PLD and the stimulation of translation by TPA, it also implies that, at best, activation of PLD is only part of the mechanism by which vasopressin increases protein synthesis in these cells. It is now clear that while PKC is involved in the stimulation of both PLD and transcription by vasopressin, the down-regulation protocol makes it possible to dissociate the two events completely. Thus, different isoforms of PKC mediate vasopressin effects on PLD and protein synthesis. Consequently, it appears that the increase in transcription we have previously observed with exogenous PLD in these cells (4Thompson M.G. Mackie S.C. Morrison K.S. Thom A. Palmer R.M. Biochim. Biophys. Acta. 1994; 1224: 198-204Crossref PubMed Scopus (14) Google Scholar) must involve an alternative mechanism to that employed by vasopressin (see below). This conclusion is also supported by the finding that the stimulation of protein synthesis at 6 h by exogenous PLD, but not vasopressin, is partially attenuated by PKC down-regulation. One possible mechanism involves the generation of lysophosphatidic acid and its action through an extracellular receptor (16Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Abstract Full Text PDF PubMed Scopus (108) Google Scholar).Immunoblotting of the down-regulated cells implicates PKC-μ, -ι, and/or -ζ in the stimulation of protein synthesis by vasopressin. However, all three isoforms have a high degree of sequence homology, raising the possibility of cross-reactivity between the antibodies. Studies have shown that the ι antibody cross-reacts with ζ, 4C. Davies, personal communication. and, since we have been unable to obtain an alternative source of ι antibody, its presence in L6 cells remains unproven at present. The antibody to PKC-μ clearly detects a protein of 115 kDa, but it fails to recognize any bands in the 70–90-kDa range. Furthermore, the antibodies to ι and ζ do not detect the 115-kDa protein. Thus, from the data available on the three atypical isoforms, it appears likely that only μ and ζ are present. The inability of TPA to down-regulate PKC μ is also of interest, since when human PKC-μ was propagated in the baculovirus expression system and purified to homogeneity, it displayed high affinity TPA binding (51Dieterich S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus (76) Google Scholar). However, this finding differs markedly from an earlier study in which only a very weak increase in TPA binding was observed in cellular extracts from PKC-μ transfectants (52Johannes F.J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar). The difference between the two observations may be due to unknown factors present in the cellular extracts that prevent TPA binding.Although the increase in translation elicited by insulin in L6 cells was not prevented by Ro-31-8220 (53Thompson M.G. Pascal M. Mackie S.C. Thom A. Morrison K.S. Backwell F.R.C. Palmer R.M. Biosci. Rep. 1995; 15: 37-46Crossref PubMed Scopus (11) Google Scholar), this inhibitor (54Thompson M.G. Acamovic F. Mackie S.C. Morrison K.S. Palmer R.M. Biosci. Rep. 1993; 13: 359-366Crossref PubMed Scopus (11) Google Scholar), but not PKC down-regulation,3 also attenuated the stimulation of transcription by insulin, suggesting that vasopressin and insulin utilize the same subset of PKC isoforms.In many of the cell lines investigated so far, PKC-ζ seems to be present as a cytosolic enzyme (e.g. see Ref. 55Crabos M. Imber R. Woodtli T. Fabbro D. Erne P. Biochem. Biophys. Res. Commun. 1991; 178: 878-883Crossref PubMed Scopus (59) Google Scholar). As predicted from its structure, most studies, including the work reported here, indicate that PKC-ζ is resistant to TPA-induced translocation or down-regulation (e.g. see Refs. 50Huwiler A. Fabbro D. Stabel S. Pfeilschifter J. FEBS Lett. 1992; 300: 259-262Crossref PubMed Scopus (59) Google Scholar and 56Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar). In addition, both of the PKC inhibitors that partially blocked the effect of vasopressin on transcription have been shown to inhibit PKC-ζ (e.g. see Ref. 57Yeo E.-J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The observation that higher concentrations of both agents (1 μm) were required to partially inhibit PKC ζ than to completely inhibit the α, δ, and ε isoforms (57Yeo E.-J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and our finding that Ro-31-8220 and bIM still elicited a partial inhibition of the vasopressin response following down-regulation further support a role for the down-regulation-resistant isoforms such as ζ in mediating the vasopressin response. In addition, vasopressin has been shown to stimulate the translocation of PKC ζ from the cytosol to a membrane fraction in human platelets (58Crabos M. Fabbro D. Stabel S. Erne P. Biochem. J. 1992; 288: 891-896Crossref PubMed Scopus (64) Google Scholar), and in smooth muscle, phenylephrine elicits PKC-ζ translocation to the nucleus (59Khalil R.A. Lajoie C. Resnick M.S. Morgan K.G. Am. J. Physiol. 1992; 263: C714-C719Crossref PubMed Google Scholar), suggesting a role in events such as gene expression.PKC-ζ has been shown to be stimulated in vitro by phosphatidylinositol 3,4,5-trisphosphate (60Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar), the phospholipid that is thought to be the physiologically important product of phosphatidylinositol-3-kinase (61Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO. J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar). However, the phosphatidylinositol-3-kinase inhibitor, wortmannin (62Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1047) Google Scholar), had no effect on the ability of vasopressin to stimulate transcription in L6 cells, suggesting that phosphatidylinositol-3-kinase is not involved in mediating this response.3 PKC-ζ is also activated by arachidonic acid (63Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar), and preliminary data show that vasopressin increases arachidonic acid release from L6 cells. 5M. G. Thompson and S. C. Mackie, unpublished observation. Furthermore, we have previously shown that vasopressin stimulates mitogen-activated protein kinase in these cells (64Thompson M.G. Mackie S.C. Thom A. Hazlerigg D.G. Morrison K.S. Palmer R.M. Biochim. Biophys. Acta. 1996; 1311: 37-44Crossref PubMed Scopus (10) Google Scholar) and mitogen-activated protein kinase is known to phosphorylate and activate cytosolic PLA2 (65Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar), releasing arachidonic acid. Thus, it is possible that vasopressin may, at least in part, stimulate protein synthesis in L6 cells through PKC-ζ via mitogen-activated protein kinase, cytosolic PLA2, and arachidonic acid. This potential sequence of events is under further investigation.Although PLD is clearly not involved in the stimulation of protein synthesis by vasopressin, both this agonist and TPA also reduce myofibrillar protein degradation in skeletal muscle (6Thompson M.G. Palmer R.M. Thom A. Garden K. Lobley G.E. Calder G. Am. J. Physiol. 1996; 270: C1875-C1879Crossref PubMed Google Scholar, 7Goodman M.N. Biochem. J. 1987; 247: 151-156Crossref PubMed Scopus (16) Google Scholar). It remains to be determined whether PLD plays a role in the regulation of this component of protein turnover. Loss of skeletal muscle is an acute metabolic response to infection and neoplastic disease and results from a decrease in the rate of protein synthesis and an increase in the rate of protein degradation (e.g. see Refs. 1Hasselgren P.O. Talamini M. James H. Fischer J.E. Arch. Surg. 1986; 121: 919-923Crossref Scopus (88) Google Scholar, 2Strelkov A.B.S. Fields A.L.A. Baracos V.E. Am. J. Physiol. 1989; 257: C261-C269Crossref PubMed Google Scholar, 3Beck S.A. Smith K.L. Tisdale M.J. Cancer Res. 1991; 51: 6089-6093PubMed Google Scholar). To reverse this process, an understanding of the signaling pathways regulating protein turnover is essential. We have used 12-O-tetradecanoylphorbol-13-acetate (TPA) 1The abbreviations used are: TPA, 12-O-tetradecanoylphorbol 13-acetate; PLD, phospholipa
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