1Diabetes Research Laboratory, Winthrop University Hospital, Mineola 11501; 3School of Medicine, State University of New York, Stony Brook, New York 11794; and 2Institut fur Klinische Biochemie und Pathobiochemie, Medizinische Universitatsklinik, D-97080 Wurzburg, Germany
Submitted 3 November 2003 ; accepted in final form 14 June 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
chemotaxis; proteasome inhibitors; mitogen-activated protein kinases; insulin; cGMP-dependent protein kinase
We recently showed (7, 18) that insulin inhibits PDGF-directed migration in VSMCs isolated from control Wistar-Kyoto (WKY) rats partly by causing dephosphorylation and inactivation of MAPKs via the induction of MKP-1, a dual-specificity tyrosine/threonine-specific phosphatase that dephosphorylates MAPK family members (32). The NO/cGMP signaling pathway mediates insulin induction of MKP-1 expression via the activation of cGMP-dependent protein kinase I (cGK I
), the downstream effector of NO/cGMP signaling (5, 18). Failure of insulin to activate NO/cGMP signaling and induce MKP-1 expression in the vasculature of diabetic rats may result in excessive VSMC growth and migration, which may contribute to enhanced atherosclerosis and restenosis observed in diabetes and hypertension.
MKP-1 and its family members are the products of immediate-early genes and therefore are under tight transcriptional control (9, 19, 20). In addition, recent evidence suggests that MKP-1 is also subjected to posttranscriptional regulation via proteasomal degradation. Thus MKP-1 is a labile protein with a half-life of 45 min and is degraded by ubiquitin-directed proteasome complex (9, 10). Furthermore, phosphorylation of MKP-1 on serine residues at the carboxy terminus is known to stabilize MKP-1 protein (9, 10). Whereas we have previously shown that insulin induces MKP-1 expression at the transcriptional level (4), it is unclear whether insulin also stabilizes MKP-1 protein.
Since diabetes is accompanied by excessive protein degradation due to defective insulin signaling, the present study was undertaken to investigate whether diabetes-related elevations in VSMC migration are mediated by impaired MKP-1 expression and stability. Furthermore, the role of cGK I in insulin-induced MKP-1 expression/stability and in diabetes was investigated with VSMCs transduced with adenovirus expressing cGK I
.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
PDGF-BB, cell culture reagents, fetal bovine serum (FBS), and antibiotics were purchased from Life Technologies (Grand Island, NY); [-32P]ATP (specific activity 3,000 Ci/mmol) from Dupont-New England Nuclear (Boston, MA); 8-bromo-cGMP (8-BrcGMP), lactacystin, MG132, and E64 from Biomol Research (Plymouth Meeting, PA); electrophoresis and protein assay reagents from Bio-Rad (Richmond, CA); type I collagenase from Worthington Biochemical (Freehold, NJ); SDS-polyacrylamide gel electrophoresis and Western blot reagents from Bio-Rad (Hercules, CA); anti-ERK2 and MKP-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); and phospho-MAPK and MAPK antibodies from New England Biolabs (Beverly, MA). cGK I antibody was prepared as described previously (18). Protein A-Sepharose CL-4B, protease inhibitors, sodium orthovanadate, and all other reagents were from Sigma (St. Louis, MO). Porcine insulin was a kind gift from Eli Lilly (Indianapolis, IN). siRNA Target Finder, Design tool, and the pSilencer siRNA expression system were purchased from Ambion (Austin, TX).
Culture of VSMCs and treatment.
VSMCs were isolated by enzymatic digestion of the aortic media of male WKY rats and Goto-Kakizaki (GK) diabetic rats weighing 200220 g, as described in our recent publications (6, 7, 18). A monoclonal antibody against smooth muscle -actin was used to assess the purity (>99%) of the SMC cultures. Unless otherwise indicated, primary cultures of VSMCs were maintained in
-MEM containing 10% FBS and 1% antibiotic/antimycotic mixture. All experiments on MAPK activation, MKP-1 expression, and cell migration were performed on confluent cells (57 days in culture) at passage 5. Before each experiment, cells were serum starved for 24 h in
-MEM and 1% antibiotics. The next day, cells were exposed to PDGF-BB (010 ng/ml), insulin (0100 nM), or insulin followed by PDGF as detailed in Figs. 110. In some experiments, VSMCs were pretreated with protease inhibitors for 6 h before insulin and subsequent exposure to PDGF, as detailed in Fig. 9, A and B.
|
|
|
Immunoblot analysis of MKP-1 and cGK I.
Confluent serum-starved VSMCs were exposed to insulin (0100 nM) for 1030 min, and equal amounts of cell lysate proteins (100 µg) were analyzed by performing Western blot analysis using either the MKP-1 or cGK I
antibody.
Immunoblot analysis of MAPK phosphorylation status. Confluent, serum-starved VSMCs treated and extracted as described above were examined using Western blot analysis. Briefly, equal amounts of proteins (2550 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, probed with phospho-p44/42 MAPK antibody or p44/42 MAPK antibody, and detected with horseradish peroxidase (HRP)-conjugated secondary antibody (18). Phospho-MAPK antibody recognizes both ERK1 and ERK2. ERK2 is the predominant isoform in VSMCs. Therefore, quantitation of the enhanced chemiluminescence (ECL) signals was performed on ERK2.
Construction of adenoviral vectors expressing MKP-1 and cGK I.
MKP-1-,
-galactosidase-, and cGK I
-expressing adenoviruses were generated by cotransfection of plasmid pACCMVpLpA-MKP-1, pACCMVpLpA-
-galactosidase, or pCMVI/cGKI
with pJM17 in HEK-293 cells as detailed in our recent publication (18). Each adenovirus was plaque purified, expanded, and titered after detection of visible plaques in a HEK-293 monolayer by agarose overlays.
Silencing of MKP-1 with siRNA strategy. Recent studies have shown that siRNA constructs targeting the 106123 region of human MKP-1 cDNA successfully blocked the expression of MKP-1 protein in BT-474 breast carcinoma cells (27). We synthesized oligonucleotides after aligning those sequences with rat MKP-1 cDNA. These oligonucleotides with overhanging ApaI and EcoRI restriction sites were cloned into the siRNA expression vector pSilencer 3.1-U6 puro, and the resulting vector was transfected into VSMCs and selected with puromycin as described previously (18). Clones were screened by Western blotting to identify those that had lowest basal as well as FBS-induced MKP-1 protein levels. One of the sequences inhibited MKP-1 expression >90%. The corresponding oligonucleotides in rat MKP-1 were 5'-CGCCGGCCACATCGTGGGC. As controls, VSMCs were transfected with pSilencer 3.1U6 puro with cloned oligonucleotides that had a composition identical to the siRNA construct but a scrambled sequence (ss) that was not complementary to any known genes.
Analysis of MKP-1 phosphorylation status. VSMCs were labeled with [32P]orthophosphate (0.5 mCi/ml) for 3 h, exposed to insulin for 0.53.0 h, followed by immunoprecipitation of 1 mg (WKY) and 4 mg (GK diabetic) lysate protein with anti-MKP-1 antibody. The immunoprecipitates were washed and separated using SDS-PAGE, and protein was transferred PVDF membrane for analysis using MKP-1 antibody.
Protein assay. Protein in cellular lysates was quantitated by the bicinchoninic acid method (28).
cGMP assay. cGMP was extracted from VSMCs with 90% ethanol. The ethanol extracts were cleared by centrifugation and evaporated, followed by reconstitution with cGMP assay buffer. cGMP was measured by using a highly sensitive radioimmunoassay kit (Amersham).
Statistics. The results are presented as means ± SE. Analysis of variance (ANOVA) followed by Dunnett's test were performed to compare the mean values between various treatments as well as control vs. GK VSMCs. A P value of <0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VSMCs isolated from GK diabetic rats exhibit elevated basal migration rate: insulin fails to inhibit VSMC migration. Our recent studies (18) showed that physiological concentrations of insulin dose-dependently inhibit PDGF-directed migration of VSMCs isolated from WKY rats. To further understand the importance and pathophysiological relevance of this inhibitory effect of insulin, we examined insulin effects on basal and PDGF-directed migration in VSMCs isolated from GK diabetic rats. As shown in Fig. 1, GK rats exhibit a 1.8-fold increase in basal VSMC migratory rates compared with control WKY rats. In WKY, PDGF caused a 2.2-fold increase in migration, whereas migration was not further increased by PDGF in GK diabetic VSMCs. Insulin treatment blocked PDGF-directed VSMC migration in WKY, but not in GK diabetic VSMCs. Notably, PDGF caused no increase in VSMC migration in GK beyond the basal values. This may be due to cellular desensitization to PDGF.
Enhanced migration in GK diabetic VSMCs is accompanied by increased MAPK phosphorylation and its activation. Numerous studies, including ours (18), have shown that MAPK signaling plays a crucial role in VSMC migration (8, 15, 21, 22). Therefore, we examined whether the elevations in basal migration observed in GK diabetic VSMCs are associated with MAPK phosphorylation. As shown in Fig. 2A, MAPK is phosphorylated in the basal state in GK diabetic but not WKY VSMCs. In WKY, PDGF treatment for 10 min resulted in a 20-fold increase in MAPK phosphorylation, which was significantly inhibited by insulin. VSMCs isolated from GK diabetic rats exhibited a 40-fold increase in PDGF-induced MAPK phosphorylation relative to basal levels. Insulin failed to inhibit PDGF-induced MAPK phosphorylation in these GK diabetic VSMCs, in contrast to a 45% decrease observed in WKY VSMCs (Fig. 2B).
|
|
|
|
|
Inhibition of MKP-1 expression through siRNA causes resistance to insulin in WKY VSMCs.
To further establish that it is indeed insulin-induced MKP-1 via cGMP/cGK I that plays a major role in insulin inhibition of PDGF-directed migration under normal conditions, MKP-1 protein expression was silenced in VSMCs derived from WKY by transfection with pSilencer-MKP-1 siRNA construct. A stable pool of VSMCs expressing the lowest basal and FBS-induced MKP-1 levels were amplified and examined for insulin effect on PDGF-directed VSMC migration. Expression of MKP-1 siRNA decreased basal and insulin-, cGMP-, cGK I
-, and proteasome inhibitor-mediated increase in MKP-1 protein expression by >90% (Fig. 7A) and blocked insulin's inhibitory effect on PDGF-mediated migration (Fig. 7B). In addition, the proteasomal inhibitor MG132 failed to prevent PDGF-directed migration in VSMCs expressing MKP-1 siRNA (Fig. 7B). Similarly, cGMP agonist as well as cGK I
did not block PDGF-directed migration in VSMCs expressing MKP-1 siRNA.
|
|
Insulin increases MKP-1 phosphorylation in WKY but not GK diabetic VSMCs. Recent evidence suggests that phosphorylation of MKP-1 stabilizes MKP-1 protein (9, 10). To investigate whether insulin treatment differentially affects MKP-1 phosphorylation, cells were metabolically labeled with [32P]orthophosphate, treated with insulin for 30 min to 3 h, and examined for MKP-1 phosphorylation status. Because GK diabetic VSMCs exhibit impaired MKP-1 protein expression, a fourfold higher amount of cell lysate proteins was used for immunoprecipitation to match the amount of MKP-1 protein immunoprecipitated from WKY VSMCs. MKP-1 phosphorylation was quantitated after normalization for MKP-1 protein by dividing the intensity of phospho-signals by the protein signals. As shown in Fig. 10, insulin caused a 2.5-fold increase in MKP-1 phosphorylation at 30 min, which was sustained for 1 h and then gradually decreased to basal levels after 3 h of insulin exposure. GK diabetic VSMCs showed reductions in basal as well as insulin-stimulated MKP-1 phosphorylation (Fig. 10).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several lines of evidence presented in this study suggest that diabetes in GK rats results in impaired basal and insulin-induced MKP-1 protein expression in VSMCs because of a combination of blunted NO/cGMP signaling and excessive proteasome degradation of the already reduced MKP-1 protein levels that allow excessive MAPK phosphorylation and VSMC migration. First, insulin-induced MKP-1 expression was markedly decreased in GK diabetic VSMCs at all concentrations of insulin tested compared with WKY VSMCs. Second, reductions in MKP-1 protein levels were accompanied by reductions in insulin-stimulated MKP-1 phosphorylation and MKP-1 enzymatic activity measured with 32P-labeled recombinant ERK2 as a substrate (data not shown). Third, decreased levels of cGK I proteins as well as decreased cGMP generation in response to insulin were observed in GK diabetic VSMCs. Fourth, restoration of MKP-1 levels by adenoviral expression in GK diabetic VSMCs decreased basal VSMC migratory rates to values comparable to those in WKY and restored responsiveness to PDGF and insulin sensitivity. Fifth, treatment with proteasome inhibitors restored MKP-1 expression in GK diabetic VSMCs to levels observed in insulin-treated WKY VSMCs and reversed diabetes-related elevations in migration. Sixth, coexpression of MKP-1 and cGK I
decreased basal and PDGF-induced MAPK phosphorylation and completely inhibited VSMC migration in GK diabetic rats under all conditions tested. Finally, siRNA-directed cellular depletion of MKP-1 in WKY VSMCs caused resistance to insulin as evidenced by a lack of insulin-dependent increase in MKP-1 expression and decrease in PDGF-mediated MAPK phosphorylation as well as migration.
Previously, we showed (24) decreased insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation in VSMCs leading to reductions in PI3-kinase enzymatic activity, iNOS protein induction, NO generation, defective insulin-stimulated relaxation, and excessive growth in GK diabetic VSMCs. The current studies add a new dimension to these findings by documenting that insulin fails to inhibit MAPK phosphorylation and migration in these VSMCs. The fact that adenoviral expression of MKP-1 only partially inhibited PDGF-induced MAPK phosphorylation in GK diabetic VSMCs whereas it completely abolished MAPK phosphorylation in WKY suggests that factors in addition to upregulation of endogenous MKP-1 levels may be necessary for dephosphorylation of MAPK and inhibition of VSMC migration. Preliminary results suggest that coexpression of cGK I along with MKP-1 indeed resulted in complete dephosphorylation and inactivation of MAPK in VSMCs isolated from GK diabetic rats. Given the knowledge that MKP-1 and its other family members associate with MAPKs and are activated by MAPKs (11, 34), it is plausible that cGK I
may facilitate the interaction between MKP-1 and MAPKs and enhance MKP-1 dephosphorylation of ERKs. Alternatively, upregulation of MKP-1 levels in GK may result in positive feedback regulation of Raf-1 and MEK as proposed by Shapiro and Ahn (26).
In addition to inducible transcription and further activation by association with MAPKs, MKP-1 is regulated by protein degradation via a ubiquitin-mediated proteasomal pathway. Moreover, phosphorylation of MKP-1 at the carboxy terminus is known to inhibit its degradation (9, 10). We did observe an increase in MKP-1 phosphorylation by insulin in WKY VSMCs, whereas those isolated from GK diabetic rats exhibited impaired insulin-mediated MKP-1 phosphorylation, which could contribute to excessive degradation of MKP-1. Thus MG132 inhibition of proteasomal degradation increased MKP-1 protein accumulation in both WKY and GK diabetic VSMCs, and caused complete reversal of migratory defects observed in GK diabetic VSMCs as well as attenuation of PDGF-directed VSMC migration in WKY. The protease inhibitor effect was specific to MKP-1, as cellular levels of cGK I and MAPKs were not altered upon treatment with MG132. Further studies are warranted to understand the role of cGK I
in MKP-1 phosphorylation.
Collectively, these findings illustrate a complex control mechanism designed to limit undesirable long-term activation of MAPKs under normal conditions and further demonstrate the importance of regulated protein degradation in the control of cell growth and migration. In contrast, in diabetes, insulin fails to inactivate MAPKs because of reduced PI3-kinase/Akt-mediated inducible nitric oxide synthase (iNOS) induction and cGMP generation, which is needed to activate cGK I, culminating in impaired MKP-1 expression. Furthermore, excessive production of growth factors observed in diabetes may interfere with insulin activation of PI3-kinase signaling and upregulate the Ras/Raf-1 signaling pathway, and thereby support increased MAPK activation and migration.
In summary, the results of the present study suggest that the diabetic condition leads to reduced cGMP and CGKK levels, leading to reduced MKP-1 expression as well as to excessive proteasomal MKP-1 degradation, all of which culminates in reduced MAPK dephosphorylation and increased VSMC migration.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This article was written in a personal capacity and does not necessarily represent the opinions or reflect the views of the National Institutes of Health, the Department of Health and Human Services, or the Federal Government.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Abedi H and Zachary I. Signalling mechanisms in the regulation of vascular cell migration. Cardiovasc Res 30: 544556, 1995.[CrossRef][ISI][Medline]
3. Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. Mol Endocrinol 14: 13651376, 2000.
4. Begum N and Ragolia L. Altered regulation of insulin signaling components in adipocytes of insulin-resistant type II diabetic Goto-Kakizaki rats. Metabolism 47: 5462, 1998.[ISI][Medline]
5. Begum N, Ragolia L, Rienzie J, McCarthy M, and Duddy N. Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells. Evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension. J Biol Chem 273: 2516425170, 1998.
6. Begum N, Sandu OA, and Duddy N. Negative regulation of rho signaling by insulin and its impact on actin cytoskeleton organization in vascular smooth muscle cells: role of nitric oxide and cyclic guanosine monophosphate signaling pathways. Diabetes 51: 22562263, 2002.
7. Begum N, Song Y, Rienzie J, and Ragolia L. Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension. Am J Physiol Cell Physiol 275: C42C49, 1998.
8. Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, and Ross R. Insulin-like growth factor-1 and platelet-derived growth factor BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest 93: 12661274, 1994.[ISI][Medline]
9. Brondello JM, Brunet A, Pouyssegur J, and McKenzie FR. The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade. J Biol Chem 272: 13681376, 1997.
10. Brondello JM, Pouyssegur J, and McKenzie FR. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 286: 25142517, 1999.
11. Camps M, Nichols A, Gilleron C, Antonsson B, Muda M, Chabert C, Boschert U, and Arkinstall S. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280: 12621265, 1998.
12. Colwell JA and Jokl R. Vascular thrombosis in diabetes. In: Diabetes Mellitus: Theory and Practice (5th ed.), edited by Porte D, Sherwin R, and Rifkin H. Norwalk, CT: Appleton and Lange, 1996.
13. Colwell JA, Lyons TJ, Klein RL, and Lopes-Virella M. New concepts about the pathogenesis of atherosclerosis and thrombosis in diabetes mellitus. In: The Diabetic Foot, edited by Levin MJ. St. Louis, MO: Mosby, 1992, p. 79114.
14. Cospedal R, Abedi H, and Zachary I. Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Cardiovasc Res 41: 708721, 1999.[CrossRef][ISI][Medline]
15. Goetze S, Xi XP, Kawano H, Gotlibowski T, Fleck E, Hsueh WA, and Law R. PPAR gamma-ligands inhibit migration mediated by multiple chemoattractants in vascular smooth muscle cells. J Cardiovasc Pharmacol 33: 798806, 1999.[CrossRef][ISI][Medline]
16. Gomez-Foix AM, Coats WS, Baque S, Alam T, Gerard RD, and Newgard CW. Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism. J Biol Chem 267: 2512925134, 1992.
17. Goto Y, Suzuki K, Sasaki M, Ono T, and Abe S. GK rat as a model of non-obese, non insulin-dependent diabetes. Selective breeding over 35 generations. In: Lessons from Animal Diabetes, edited by Shafrir E and Renold AE. London: Smith-Gordon, 1988, vol. 2, p. 107116.
18. Jacob A, Molkentin JD, Smolenski A, Lohmann SM, and Begum N. Insulin inhibits PDGF-directed VSMC migration via NO/cGMP increase of MKP-1 and its inactivation of MAPK. Am J Physiol Cell Physiol 283: C704C713, 2002.
19. Kamps M, Nichols A, and Arkinstall S. Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J 14: 616, 2000.
20. Keyse SM. Protein phosphatases and the regulation of mitogen-activated protein kinase signaling. Curr Opin Cell Biol 12: 186192, 2000.[CrossRef][ISI][Medline]
21. Lundberg MS, Curto KA, Bilato C, Monticone RE, and Crow MT. Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways. J Mol Cell Cardiol 30: 23772389, 1998.[CrossRef][ISI][Medline]
22. Nelson PR, Yamamura S, Mureebe L, Itoh H, and Kent C. Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase. J Vasc Surg 27: 117125, 1998.[ISI][Medline]
23. Ostenson CG, Khan A, Abdel-Halim SM, Guenifi A, Suzuki KI, Goto Y, and Efendic S. Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat. Diabetologia 36: 38, 1993.[ISI][Medline]
24. Sandu OA, Ragolia L, and Begum N. Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation. Diabetes 49: 21782189, 2000.[Abstract]
25. Schwartz SM. Perspectives series: cell adhesion in vascular biology. Smooth muscle migration in atherosclerosis and restenosis. J Clin Invest 99: 28142816, 1997.
26. Shapiro PS and Ahn NG. Feedback regulation of Raf-1 and mitogen activated protein kinase (MAP) kinase kinases 1 and 2 by MAPK kinase phosphatase-1 (MKP-1). J Biol Chem 273: 17881793, 1998.
27. Small GW, Somasundaram S, Moore DT, Shi YY, and Orlowski RZ. Repression of mitogen-activated protein kinase (MAPK) phosphatase-1 by anthracyclines contributes to their antiapoptotic activation of p42/44-MAPK. J Pharmacol Exp Ther 307: 861869, 2003.
28. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150: 7685, 1985.[ISI][Medline]
29. Sohaskey ML and Ferrell JE Jr. Activation of p42 mitogen-activated protein kinase (MAPK), but not c-Jun NH2-terminal kinase, induces phosphorylation and stabilization of MAPK phosphatase XCL100 in Xenopus oocytes. Mol Biol Cell 13: 454468, 2002.
30. Sowers JR, Epstein M, and Frolich ED. Diabetes, hypertension, and cardiovascular disease: an update. Hypertension 37: 10531059, 2001.
31. Stout RW. Insulin and atherogenesis. Eur J Epidemiol 8: 134135, 1992.[ISI][Medline]
32. Takehara N, Kawabe J, Aizawa Y, Hasebe N, and Kikuchi K. High glucose attenuates insulin-induced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in vascular smooth muscle cells. Biochim Biophys Acta 1497: 244252, 2000.[CrossRef][ISI][Medline]
33. Vaandrager AB, Tilly BC, Smolenski A, Schneider-Rasp S, Bot AG, Edixhoven M, Scholte BJ, Jarchau T, Walter U, Lohmann SM, Poller WC, and de Jonge HR. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta. J Biol Chem 272: 41954200, 1997.
34. Zhou B, Wu L, Shen K, Zhang J, Lawrence DS, and Zhang ZY. Multiple regions of MAPK phosphatase are involved in its recognition and activation by ERK2. J Biol Chem 276: 65066515, 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |