RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions.
The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.
Department of Pharmacology and the Center for Developmental Genetics, State University of New York at Stony Brook, Stony Brook, NY 11794-5140, USA.
Wenjuan
W
Su
Pierre
P
Chardin
Masakazu
M
Yamazaki
Yasunori
Y
Kanaho
Guangwei
G
Du
D002460:Cell Line; D022001:Focal Adhesions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D022502:Stress Fibers / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020742:rhoA GTP-Binding Protein / Q000378:metabolism* / Q000494:pharmacology
D010739:Phospholipase D; C106801:phospholipase D1; D020742:rhoA GTP-Binding Protein