Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation.
We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.
Garnovskaya
Maria N
MN
Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center and Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina 29425, USA. garnovsk@musc.edu
Mukhin
Yurii V
YV
Vlasova
Tamara M
TM
Grewal
Jasjit S
JS
Ullian
Michael E
ME
Tholanikunnel
Baby G
BG
Raymond
John R
JR
D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015151:Immunoblotting; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008934:Mitogens / Q000378:metabolism*; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins; D051381:Rats; D016160:Retinoblastoma Protein / Q000235:genetics*; D012694:Serine / Q000737:chemistry*; D012701:Serotonin / Q000378:metabolism; D013997:Time Factors; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D008934:Mitogens; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D000804:Angiotensin II; D019938:Cyclin D1; D012701:Serotonin; D012694:Serine; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin