A new paxillin-binding protein, PAG3/Pap alpha/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration

Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is loc alized to focal contacts but seems to also exist in a relatively large cyto plasmic pool. Here, we report the identification of a new paxillin-binding protein, FAGS (paxillin-associated protein with ADP-ribosylation factor [AR F] GTPase-activating protein [GAP] activity, number 3), which is involved i n regulation of the subcellular localization of paxillin. FAGS bound to all paxillin isoforms and was induced during monocyte maturation, at which tim e paxillin expression is also increased and integrins are activated. PAG3 w as diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at f ocal adhesion plaques, suggesting that PAG3 is not an integrin assembly pro tein. PAG3 was identical to KIAA0400/Pap alpha, which was previously identi fied as a Pyk2-binding protein bearing a GAP activity toward several ARFs i n vitro. Mammalian ARFs fall into three classes, and we showed that all cla sses could affect subcellular localization of paxillin. We also examined po ssible interaction of FAGS with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of F AGS. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inh ibited paxillin recruitment to focal contacts and hampered cell migratory a ctivities, whereas cell adhesion activities were almost unaffected. Therefo re, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be i nvolved in this process, possibly through its GAP activity toward ARF prote ins. Our result thus delineates a new aspect of regulation of cell migrator y activities.