Mitogen-activated protein (MAP) kinase is regulated by the MAP kinase phosphatase (MKP-1) in vascular smooth muscle cells. Effect of actinomycin D and antisense oligonucleotides.
Angiotensin II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC) and activates many growth-promoting kinases such as mitogen-activated protein (MAP) kinase. A novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1), is induced by angiotensin II in VSMC and selectively dephosphorylates MAP kinase in vitro. Using actinomycin D and antisense oligonucleotides targeted to MKP-1, we demonstrate that MKP-1 regulates MAP kinase in VSMC. Both actinomycin D and MKP-1 antisense oligonucleotides inhibited MKP-1 mRNA expression and caused prolonged activation of the p42 and p44 MAP kinases as measured by in-gel-kinase assays and Western blot. For example, MAP kinase activity 120 min after angiotensin II treatment was 30% (range 25-35%), 79%, and 74% of maximum in control, actinomycin D-treated (3 micrograms/ml, 30 min), and antisense oligonucleotide-treated (300 nM, 6 h) cells, respectively. A sense oligonucleotide was without effect (34%). MKP-1 antisense oligonucleotides did not affect the activity of MEK indicating that sustained activation of MAP kinase was due to inhibition of MKP-1 expression. These findings demonstrate that inactivation of MAP kinase by angiotensin II is mediated predominantly by MKP-1, suggesting an important role for MKP-1 and other related phosphatases in the regulation of MAP kinases in VSMC.
Duff
J L
JL
Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA.
Monia
B P
BP
Berk
B C
BC
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins; D003609:Dactinomycin / Q000494:pharmacology*; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation; D004794:Enzyme Repression / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects*; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D063086:Organothiophosphates; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D016376:Oligonucleotides, Antisense; D063086:Organothiophosphates; D012333:RNA, Messenger; D000804:Angiotensin II; D003609:Dactinomycin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases