Recruitment of protein phosphatase 2A to dorsal ruffles by platelet-derived growth factor in smooth muscle cells: dephosphorylation of Hsp27.
In this study, we investigated the mechanism underlying Hsp27 dephosphorylation in smooth muscle cells. We found that protein phosphatase 2A (PP2A) dephosphorylates Hsp27. In addition, Hsp27 dephosphorylation was regulated by membrane cholesterol content. We showed that PDGF induced a three-fold increase in the proportion of PP2A activity regulated by cholesterol in the Triton-insoluble fraction of cell lysates. Moreover, cholesterol depletion decreased the amount of PP2A recovered in Triton-insoluble fraction. Thus, PDGF might regulate a small pool of PP2A associated with lipid rafts. Isolation of detergent-resistant membrane fragments by Optiprep-gradient density indicated that this pool of PP2A was not associated with caveolae, but was recovered in a higher density fraction (DRM-H) with ganglioside GM1, alpha-actinin, Hsp27 and p34, a component of Arp2/3 complex. These proteins were also present in dorsal ruffles containing GM1 but not caveolin-1. Phosphorylated Hsp27 levels detected in dorsal ruffles were variable. Cholesterol depletion, which inhibits dorsal ruffle formation, decreased PP2A levels and increased the Hsp27-P to Hsp27 ratio in DRM-H. These findings suggest that Hsp27 is dephosphorylated by PP2A in dorsal ruffles, in non-caveolar lipid raft microdomains. However, similarly to p34, non-phosphorylated Hsp27 is associated to non-raft membrane domains at the leading edge of lamellipodia.
Berrou
Eliane
E
Centre de Recherche Cardiovasculaire INSERM Lariboisière, U689-E4, Hôpital Lariboisière, 8 rue Guy Patin, 75010 Paris, France. eliane.berrou@inserm.fr
Bryckaert
Marijke
M
D000818:Animals; D021941:Caveolae / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism* / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism*; D013552:Swine; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D055551:HSP27 Heat-Shock Proteins; D010982:Platelet-Derived Growth Factor; D002784:Cholesterol; D048051:p38 Mitogen-Activated Protein Kinases; D054648:Protein Phosphatase 2