Mechanism of platelet-derived growth factor-dependent caveolin-1 phosphorylation: relationship to sterol binding and the role of serine-80.
In human vascular smooth muscle cells, inhibitors of protein kinase C activity reduced serine phosphorylation of caveolin-1 and increased sterol binding by this protein. This was measured after immunoprecipitation of caveolin-1 from cells labeled with tritiated cholesterol or the photoactivable cholesterol analogue FCBP [Fielding et al. (2002) Biochemistry 41, 4929-4937]. At the same time cellular sterol efflux was inhibited. Mutagenesis within a caveolin-1 central domain (residues 80-104) suggested a major role for serine-80 in mediating both of these effects. To perturb sterol binding, platelet-derived growth factor was added to the cells, leading to a transient loss of caveolin-1-associated sterol. Under these conditions, sterol efflux was stimulated, and caveolin-1 phosphorylation at tyrosine(14), assayed with a selective antibody, was substantially increased above baseline levels. These changes were also blocked by inhibitors of protein kinase C activity. Selective inhibitors of the platelet-derived growth factor receptor and downstream kinases were used to show that loss of sterol from caveolin-1 preceded tyrosine phosphorylation, but relipidation was dependent on phosphotyrosine hydrolysis.
Cardiovascular Research Institute and Departments of Medicine, University of California, San Francisco, California 94143, USA. pefield@itsa.ucsf.edu
Phoebe E
PE
Fielding
Phuonglan
P
Chau
Dong
D
Liu
Thomas A
TA
Spencer
Christopher J
CJ
Fielding
D000924:Anticholesteremic Agents / Q000737:chemistry; D001692:Biological Transport; D021941:Caveolae / Q000378:metabolism / Q000502:physiology; D051242:Caveolin 1; D022461:Caveolins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000737:chemistry; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014443:Tyrosine / Q000096:biosynthesis / Q000235:genetics
D000924:Anticholesteremic Agents; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D009842:Oligopeptides; D010455:Peptides; D010982:Platelet-Derived Growth Factor; D014443:Tyrosine; D012694:Serine; D002784:Cholesterol; C085706:FLAG peptide; D017479:Receptors, Platelet-Derived Growth Factor; D011493:Protein Kinase C