1Cardiovascular Sciences and 2Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208
Submitted 15 May 2003 ; accepted in final form 23 January 2004
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ABSTRACT |
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protein kinase C-; calcium/calmodulin-dependent protein kinase II-
2; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus
Recent studies have linked GPCR-stimulated pathways with growth factor receptor-dependent activation of ERK1/2 through a pathway involving EGF receptor (EGFR) transactivation (22). This occurs either through direct phosphorylation of the EGFR by src, which, in turn, triggers EGFR autophosphorylation necessary for interaction with adaptor proteins (30), or a metalloprotease-dependent release of membrane-bound heparin-binding EGF, which then acts as a ligand for the EGFR (10). The nonreceptor tyrosine kinase proline-rich tyrosine kinase (PYK2) may also be a component of this EGFR transactivation pathway (34). PYK2 is known to be activated by Ca2+-dependent or PKC-dependent pathways (2).
It is not fully understood how GPCR agonists couple to the activation of nonreceptor tyrosine kinases such as the src family kinases (SFKs) or PYK2. One potential mechanism is via GPCR-dependent activation of PLC and subsequent generation of diacylglycerol (DAG) and inositol trisphosphate, leading to PKC activation and increased intracellular free Ca2+ concentration, respectfully. In VSM cells, angiotensin II has been reported to result in EGFR transactivation and ERK1/2 activation by activating nonreceptor tyrosine kinases by a PKC-dependent mechanism (11). Recently, we reported evidence that Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates PYK2 and ERK1/2 activation in response to Ca2+-mobilizing stimuli in VSM cells and that ERK1/2 activation under these circumstances required a SFK and EGFR transactivation (15). Thus activation of PLC results in the generation of two intracellular signals (DAG and Ca2+) that can independently activate nonreceptor tyrosine kinases, EGFR transactivation, and ERK1/2 activation through the actions of intermediate multifunctional serine/threonine kinases. An alternative pathway has also been described that involves G protein -subunit-mediated scaffolding of adaptor proteins and ras-activating proteins (32).
On the basis of knowledge that multiple signaling pathways may impinge on nonreceptor tyrosine kinases and ultimately ERK1/2, it appears likely that utilization of these pathways would be stimulus, cell type, and situation dependent. To discern the relative contribution of a given input pathway, experimental approaches capable of acutely inhibiting the activity of endogenous components are required. In the case of PKC-dependent signaling, this approach is confounded by the fact that PKCs are a large family of protein kinases consisting of conventional (cPKC) isozymes (,
I,
II, and
) that require Ca2+ and DAG for activation, novel (nPKC) isozymes (
,
,
,
, µ) that require only DAG, and atypical PKCs (
and
/
) with mechanisms of activation that are Ca2+ and DAG independent (35). We have previously reported that PKC-
and -
are abundant isozymes in cultured VSM cells (35) and that suppression of PKC-
expression with the use of antisense oligonucleotides had no effect on phorbol 12,13-dibutyrate (PDBu)-induced ERK1/2 activation in VSM cells (7). On this basis, we have hypothesized that PKC-
mediates PDBu-induced ERK1/2 activation in VSM and propose that this would also extend to GPCR-induced ERK1/2 responses.
In the present study, we used specific pharmacological and molecular approaches to 1) assess the relative contribution of CaMKII- and PKC--dependent pathways in mediating ATP-stimulated ERK1/2 and 2) determine the relative contributions of PKC-
and PKC-
to phorbol ester- and ATP-stimulated ERK1/2 activation in cultured VSM cells. The results indicate important roles for both PKC and CaMKII as intermediates linking a GPCR agonist (ATP) to activation of tyrosine kinases and ras-dependent signaling pathways leading to ERK1/2 activation in VSM cells. Furthermore, we found that the PKC-dependent pathway mediating PDBu- and ATP-dependent ERK1/2 in VSM cells is specifically mediated by PKC-
.
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METHODS |
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Immunoprecipitations and Western blotting. Cells were lysed (0.5 ml/60 mm dish or 1 ml/100 mm dish) in a modified RIPA buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 10% glycerol, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 0.2 U/ml aprotinin). After lysis, the samples were centrifuged to clear the lysate of the insoluble debris and preincubated with 20 µg protein A beads by rocking for 30 min at 4°C, followed by centrifugation and transfer to a fresh 1.5-mm tube. Primary antibody was incubated for 90 min before addition of 20 µg of protein A beads to capture the immune complexes. The pelleted beads were then washed three times with 0.5-ml RIPA buffer, dissolved in 3x SDS-PAGE sample buffer, and heated for 5 min at 95°C. Samples were resolved by using standard SDS-PAGE procedures, transferred to nylon-backed nitrocellulose (MSI), and immunoblotted. Following blocking in 5% nonfat dry milk or 3% BSA, the immunoblots were incubated for either 1 h at room temperature or overnight at 4°C, washed three times for 10 min with TBST (20 mM Tris, 150 mM NaCl, 0.2% Tween 20), and incubated for 1 h with appropriate secondary antibody (horseradish peroxidase conjugate; Amersham). The blots were then washed three times for 10 min with TBST, incubated in enhanced chemiluminescent substrate (Amersham), and exposed to X-ray film (E. M. Parker).
Generation of dominant-negative ras adenovirus. An adenovirus containing an NH2-terminal hemagglutinin-tagged dominant-negative ras construct (Ad.HA-RasN17) was constructed by using the AdEasy system, as previously described (27). This viral construct also encoded green fluorescent protein (GFP) under a separate cytomegalovirus promoter. Experiments were typically performed by using a multiplicity of infection (MOI) of 20, and under these conditions nearly 100% of the cells were infected, as determined by the visualization of the coexpressed GFP in the recombinant virus (not shown).
Cloning and generation of CaMKII-2 adenovirus.
Mutations to CaMKII-
2 were engineered by using the Transformer Site Directed Mutagenesis kit (Clontech, Palo Alto, CA). Kinase-negative (KN)-CaMKII-
2 was generated by replacement of the lysine 43 with an alanine (K43A). Adenoviral stocks encoding KN-CaMKII-
2 were produced in collaboration with Dr. Michael Crow (Johns Hopkins, Baltimore, MD). Adenovirus encoding
-galactosidase (Ad.LacZ) was a gift from Dr. Rebecca Keller (Albany Medical College). All adenovirus stocks were propagated by adding small amounts of virus to human embryonic kidney-293 cells. When cells were
50% lysed, cells along with media were collected, subjected to three freeze-thaw cycles, aliquoted, and stored at 80°C. Titer assays were performed by the method of O'Carroll et al. (29). All assays were performed by using
-galactosidase as an adenoviral control at matching MOI.
PKC activity assay.
PKC- and PKC-
were immunoprecipitated from VSM cells and assayed as described earlier (23). After being washed three times with immunoprecipitation buffer and once in sucrose buffer (10 mM MOPS, pH 7.4, 250 mM sucrose, 2.5 mM EGTA, 2 mM EDTA, 0.2 U/ml aprotinin, and 0.2 mM phenylmethylsulfonyl fluoride), the protein A beads were incubated for 10 min at 30°C in 50 mM HEPES, pH 7.4, 10 mM Mg(Ac)2, 2 mM CaCl2, 1 mM EGTA, 0.2 mg/ml histone IIIs, 1.4 µg/µl phosphatidyl serine, 0.2 µg/µl diolein, 1 mM ATP, and 2 µCi/reaction [32P]ATP. After incubation, 25 µl of reaction were spotted onto P81 filter paper and washed five times in 75 mM phosphoric acid and once in ethanol. After drying, 32P incorporation was determined by scintillation counting by using a Beckman LS6500 scintillation counter.
Materials.
KN-PKC- (AdKN-PKC-
) and KN-PKC-
adenovirus (AdKN-PKC-
) were a gift from Dr. Trevor Biden (Baker Heart Research Institute, Melbourne, Australia). PKC-
and PKC-
were rendered kinase negative by a point mutation in the ATP-binding region of their kinase domain, and replication-deficient adenoviruses were generated as previously described (8).
Polyclonal antibodies to PKC-, PKC-
, and PKC-
were purchased from Santa Cruz (Santa Cruz, CA). Monoclonal antibodies for PKC-
, PKC-
, and ERK2 were purchased from Transduction Laboratories (Lexington, KY). The antibodies specific for active and total ERK1/2 and phosphorylated PKC (Ser660) were purchased from Cell Signaling Technology (Beverly, MA). All tissue culture media were purchased from GIBCO-BRL (Life Technologies), unless specifically stated. Tissue culture supplies (dishes, pipettes, etc.) were purchased from Fisher Scientific. Protein A-coated beads were purchased from Pierce SDS-PAGE, and Western blotting supplies were purchased from Bio-Rad, unless otherwise stated. All other chemicals were purchased from Sigma (St. Louis, MO).
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RESULTS |
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DISCUSSION |
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In recent studies, by using pharmacological approaches similar to those used here, it was concluded that PKC- mediated ERK1/2 activation in response to stimulation by angiotensin II in rat liver epithelial cells (24) and ERK1/2 activation and prostacyclin synthesis in endothelial cells in response to VEGF (16). On the other hand, studies using cardiomyocytes indicated that overexpression of a constitutively active PKC-
mutant resulted in only small increases in ERK1/2 activation compared with constitutively active PKC-
(21). In contrast, overexpression of constitutively active PKC-
was found in that study to induce large increases in c-Jun NH2-terminal kinase and p38MAPK activation. PKC-
has also been implicated in c-Jun NH2-terminal kinase activation in response to genotoxic agents in human U-937 myeloid leukemia cells (41). On the basis of the current study and literature, it is apparent that PKC isozymes can selectively couple to MAPK signaling pathways, although the particular isozymes and pathways may be cell-type specific.
The conclusions here rely, in part, on the specificities of PKC inhibitors, which have been questioned (9). However, control experiments indicated that rottlerin and Gö-6976 selectively inhibited PKC- and PKC-
autophosphorylation and in vitro kinase activity, respectively, clearly establishing their specificity relative to each other. Furthermore, the lack of effect of these inhibitors on ERK1/2 activation in response to other stimuli that do not depend on PKC (i.e., EGF, Iono) strongly suggests a lack of nonspecific effects of these agents on numerous intermediate scaffolding steps and protein kinases in the ERK activation cascade. Finally, the results obtained from overexpression of the KN-PKC-
and -PKC-
constructs fully support the pharmacological approach and establish a selective function for PKC-
in ERK1/2 activation in response to either PDBu or ATP.
Involvement of ras as an intermediate in PKC-dependent ERK1/2 activation stimulated by phorbol esters or GPCR agonists has been studied but is still a matter of controversy. There are a number of reports that conclude that ras-dependent pathways are not involved in, or only partially account for, ERK1/2 activation. Generally, the data supporting this conclusion are low levels of measurable ras activation stimulated by phorbol ester activators of PKC or stimuli acting through GPCRs and/or incomplete inhibition of ERK1/2 activation in response to these stimuli in cells overexpressing dominant-negative ras constructs. For example, angiotensin II-stimulated ERK activation in GN4 rat epithelial cells (24) and VSM cells (36) has been reported to be incompletely inhibited by overexpression of RasN17. In the present study, PDBu- and ATP-induced ERK1/2 activation was partially inhibited by overexpressing HA-RasN17, whereas EGF-induced ERK activation was completely blocked by this treatment. This result clearly indicates the efficacy of the RasN17 overexpression approach and supports the idea that at least part of the PDBu- and ATP-dependent activation of ERK1/2 in these cells is ras dependent. Similar conclusions have been reached by regarding a positive role for ras-dependent pathways in mediating angiotensin II-stimulated ERK1/2, Akt, and p70S6K activities (12).
To determine whether incomplete inhibition of PDBu- and ATP-dependent responses by overexpression of RasN17 reflects involvement of ras-independent pathways requires further investigation. Interestingly, overexpression of constitutively active PKC-, but not PKC-
or PKC-
, has been reported to activate MEK1 and ERK1 in COS and NIH/3T3 cells, a response dependent on c-raf but only partially inhibited by RasN17 (37). These results in transformed cell lines, and the present results in primary cultures of VSM cells using approaches that modify endogenous PKC
activity, suggest that PKC-
may activate ras-independent pathways leading to ERK, perhaps by directly activating raf. Raf has been shown to directly interact with several PKC isozymes, including PKC-
(16), -
(13), and -
(38), and specific PKC phosphorylation sites have been identified in raf itself (38). However, phosphorylation of these sites alone is not sufficient to activate raf (25). PKC-
-raf interactions appear to require 14-3-3 proteins as intermediate binding proteins (38), and it has been recently reported that 14-3-3 proteins are necessary for complete activation of raf (40). It is plausible that ras-independent activation of ERK1/2 involving PKC-
may depend on similar interactions with raf, mediated by 14-3-3 proteins.
In addition to activation of PKC, many GPCR agonists stimulate increases in free intracellular Ca2+ in VSM cells, leading to activation of Ca2+-dependent signaling pathways, including multifunctional Ca2+/calmodulin-dependent protein kinases. In previous studies, we have implicated CaMKII as an important mediator of ERK1/2 activation in response to Ca2+-dependent stimuli in VSM cells. Similar conclusions have been reached in other cell systems, including PC12 cells and endothelial cells. However, all of the studies to date have been indirect and have relied on pharmacological approaches using calmodulin antagonists and CaMKII-selective inhibitors (KN-62, KN-93) and/or by overexpressing constitutively active CaMKII subunits. Recently, we have characterized the consequences of overexpressing a KN-CaMKII-2 construct in VSM cells and demonstrated that it functions as an inhibitor of endogenous CaMKII-
2 activity with respect to substrate phosphorylation, both in vitro and in the intact cell (31). In the present study, we provide the first evidence using this dominant-negative approach to demonstrate involvement of CaMKII-
2 in GPCR agonist-stimulated ERK1/2 activation.
On the basis of the ras dependency for Iono-dependent activation of ERK1/2 shown in this study and previous results indicating CaMKII-dependent activation of nonreceptor tyrosine kinases and EGFR transactivation, it appears that Ca2+ and PKC--dependent signals activate common pathways leading to stimulation of ERK1/2. The combined contributions of PKC-
and CaMKII-
2 nearly completely account for ATP-induced activation of ERK1/2 and suggest a common downstream target at the level of the nonreceptor tyrosine kinases, as indicated in the proposed model (Fig. 10). This model is consistent with previous studies implicating nonreceptor tyrosine kinases and EGFR transactivation as intermediates in GPCR agonist stimulation of ERK1/2 in VSM cells (12, 34). However, the indirect pharmacological approaches used here to implicate involvement of nonreceptor tyrosine kinases do not rule out the possibility that other independent mechanisms may be involved in activating the EGFR receptor (10). Additional studies are warranted to determine the mechanisms by which PKC-
and CaMKII-
2 regulate nonreceptor tyrosine kinases and/or EGFR transactivation, as well as the functional implications of PKC-
- and CaMKII-
2-dependent ERK1/2 activation in response to ATP and other GPCR agonists.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Avraham H, Park SY, Schinkmann K, and Avraham S. RAFTK/Pyk2-mediated cellular signalling. Cell Signal 12: 123133, 2000.[CrossRef][ISI][Medline]
3. Beltman J, McCormick F, and Cook SJ. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem 271: 2701827024, 1996.
4. Berk BC. Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 81: 9991030, 2001.
5. Bornfeldt KE, Campbell JS, Koyama H, Argast GM, Leslie CC, Raines EW, Krebs EG, and Ross R. The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets. J Clin Invest 100: 875885, 1997.
6. Burnstock G. Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 22: 364373, 2002.
7. Busuttil SJ, Morehouse DL, Youkey JR, and Singer HA. Antisense suppresssion of protein kinase C-alpha and -delta in vascular smooth muscle. J Surg Res 63: 137142, 1996.[CrossRef][ISI][Medline]
8. Carpenter L, Cordery D, and Biden TJ. Inhibition of protein kinase C delta protects rat INS-1 cells against interleukin-1 beta and streptozotocin-induced apoptosis. Diabetes 51: 317324, 2002.
9. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95105, 2000.[CrossRef][ISI][Medline]
10. Eguchi S, Dempsey PJ, Frank GD, Motley ED, and Inagami T. Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK. J Biol Chem 276: 79577962, 2001.
11. Eguchi S, Iwasaki H, Inagami T, Numaguchi K, Yamakawa T, Motley ED, Owada KM, Marumo F, and Hirata Y. Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension 33: 201206, 1999.
12. Eguchi S, Iwasaki H, Ueno H, Frank GD, Motley ED, Eguchi K, Marumo F, Hirata Y, and Inagami T. Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt. J Biol Chem 274: 3684336851, 1999.
13. Formisano P, Oriente F, Fiory F, Caruso M, Miele C, Maitan MA, Andreozzi F, Vigliotta G, Condorelli G, and Beguinot F. Insulin-activated protein kinase C beta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells. Mol Cell Biol 20: 63236333, 2000.
14. Geisterfer AA, Peach MJ, and Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 62: 749756, 1988.[Abstract]
15. Ginnan R and Singer HA. CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle. Am J Physiol Cell Physiol 282: C754C761, 2002.
16. Gliki G, Abu-Ghazaleh R, Jezequel S, Wheeler-Jones C, and Zachary I. Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-delta and by mobilization of intracellular Ca2+. Biochem J 353: 503512, 2001.[CrossRef][ISI][Medline]
17. Griendling KK, Ushio-Fukai M, Lassegue B, and Alexander RW. Angiotensin II signaling in vascular smooth muscle. New concepts. Hypertension 29: 366373, 1997.
18. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, and Johannes FJ. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett 392: 7780, 1996.[CrossRef][ISI][Medline]
19. Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, and Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199: 9398, 1994.[CrossRef][ISI][Medline]
20. Hauck CR, Hsia DA, and Schlaepfer DD. Focal adhesion kinase facilitates platelet-derived growth factor-BB-stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells. J Biol Chem 275: 4109241099, 2000.
21. Heidkamp MC, Bayer AL, Martin JL, and Samarel AM. Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes. Circ Res 89: 882890, 2001.
22. Kalmes A, Daum G, and Clowes AW. EGFR transactivation in the regulation of SMC function. Ann NY Acad Sci 947: 4254, 2001.
23. Li W, Yu JC, Shin DY, and Pierce JH. Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J Biol Chem 270: 83118318, 1995.
24. Li X, Lee JW, Graves LM, and Earp HS. Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway. EMBO J 17: 25742583, 1998.
25. Marais R, Light Y, Mason C, Paterson H, Olson MF, and Marshall CJ. Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280: 109112, 1998.
26. McNamara CA, Sarembock IJ, Gimple LW, Fenton JW, Coughlin SR, and Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 91: 9498, 1993.[ISI][Medline]
27. Meadows KN, Bryant P, and Pumiglia K. Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation. J Biol Chem 276: 4928949298, 2001.
28. Newton AC. Protein kinase C: structure, function and regulation. J Biol Chem 270: 2849528498, 1995.
29. O'Carroll SJ, Hall AR, Myers CJ, Braithwaite AW, and Dix BR. Quantifying adenoviral titers by spectrophotometry. Biotechniques 28: 408412, 2000.[ISI][Medline]
30. Parsons JT and Parsons SJ. Src family protein tyrosine kinases: cooperating with growth factor and adhesion signaling pathways. Curr Opin Cell Biol 9: 187192, 1997.[CrossRef][ISI][Medline]
31. Pfleiderer PJ, Lu KK, Crow MT, Keller RS, and Singer HA. Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-2. Am J Physiol Cell Physiol 286: C1238C1245, 2004.
32. Pumiglia KM, LeVine H, Haske T, Habib T, Jove R, and Decker SJ. A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase. J Biol Chem 270: 1425114254, 1995.
33. Robinson MJ and Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9: 180186, 1997.[CrossRef][ISI][Medline]
34. Shah BH and Catt KJ. Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: roles of protein kinase C delta, Src/proline-rich tyrosine kinase 2, and epidermal growth receptor trans-activation. Mol Pharmacol 61: 343351, 2002.
35. Singer HA. Protein kinase C. In: Biochemistry of Smooth Muscle Contraction, edited by Bárány M. San Diego, CA: Academic, 1996, p. 155166.
36. Takahashi T, Kawahara Y, Okuda M, Ueno H, Takeshita A, and Yokoyama M. Angiotensin II stimulates mitogen-activated protein kinases and protein synthesis by a Ras-independent pathway in vascular smooth muscle cells. J Biol Chem 272: 1601816022, 1997.
37. Ueda Y, Hirai S, Osada S, Suzuki A, Mizuno K, and Ohno S. Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf. J Biol Chem 271: 2351223519, 1996.
38. Van Der Hoeven PC, Van Der Wal JC, Ruurs P, Van Dijk MC, and Van Blitterswijk J. 14-3-3 Isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation. Biochem J 345: 297306, 2000.[CrossRef][ISI][Medline]
39. Van Riper DA, Schworer CM, and Singer HA. Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle. Mol Cell Biochem 213: 8392, 2000.[CrossRef][ISI][Medline]
40. Yip-Schneider MT, Miao W, Lin A, Barnard DS, Tzivion G, and Marshall MS. Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association. Biochem J 351: 151159, 2000.[CrossRef][ISI][Medline]
41. Yoshida K, Miki Y, and Kufe D. Activation of SAPK/JNK signaling by protein kinase C delta in response to DNA damage. J Biol Chem 277: 4837248378, 2002.