title: Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein.
abstract: FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.
authors.affiliation: From the Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Minami-ku, Kanagawa 252-0373, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Morishita
authors.affiliation: From the Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Minami-ku, Kanagawa 252-0373, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Tsutsumi
authors.affiliation: From the Division of Cell Biology, Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Minami-ku, Kanagawa 252-0373, Japan yohta@kitasato-u.ac.jp.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Ohta
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D034622:RNA Interference; D012694:Serine / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000089982:ADP-Ribosylation Factor 6; C497033:ARHGAP24 protein, human; D020690:GTPase-Activating Proteins; D012694:Serine; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human
keywords: Rac (Rac GTPase); Rho (Rho GTPase); actin; cell adhesion; phosphorylation; signal transduction; small GTPase