A PLCdelta1-binding protein, p122/RhoGAP, is localized in caveolin-enriched membrane domains and regulates caveolin internalization.
A GTPase activating protein (GAP), p122, has previously been cloned as a phospholipase C (PLC)delta1-interacting protein. p122 shows a specific GAP activity for Rho and enhances the enzyme activity of PLCdelta1. In this study, we examined the localization and functions of p122/RhoGAP, using enhanced green fluorescent protein (EGFP)-tagged proteins. EGFP-p122 was observed as punctate structures at the plasma membrane of BHK (fibroblastic) cells and MDCK (epithelial) cells. This patchy distribution depended on membrane cholesterol levels and the C-terminal region of p122 containing the GAP domain was responsible for it. Sucrose density gradient centrifugation and immunostaining of caveolin-1 revealed that p122 was localized in caveolin-enriched membrane domains mainly via its GAP domain. We demonstrated that transient expression of EGFP-p122 caused internalization of caveolin-1. Moreover, when the EGFP-tagged GAP domain was introduced in another fibroblastic cell line, NRK cells, punctate fluorescent structures were co-localized with caveolin-1. In this case, caveolin-1-positive structures were found in patches of F-actin, unlike those of untransfected cells that formed linear arrays along with actin stress fibres. These results suggest that p122 is localized in caveolae and plays an important role in caveolin distribution through reorganization of the actin cytoskeleton.
Yamaga
Masaki
M
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678-1297, Japan.
Sekimata
Masayuki
M
Fujii
Makoto
M
Kawai
Katsuhisa
K
Kamata
Hideaki
H
Hirata
Hajime
H
Homma
Yoshimi
Y
Yagisawa
Hitoshi
H
D000199:Actins / Q000378:metabolism; D000818:Animals; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008856:Microscopy, Fluorescence
D000199:Actins; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D020690:GTPase-Activating Proteins; D002784:Cholesterol