A New Paxillin-binding Protein, PAG3/Papα/KIAA0400, Bearing an ADP-Ribosylation Factor GTPase-activating Protein Activity, Is Involved in Paxillin Recruitment to Focal Adhesions and Cell Migration

Abstract

Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, PAG3 (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. PAG3 bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was 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 focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Papalpha, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of PAG3 with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of PAG3. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities.

Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells

“…Rac1 activity is regulated by GEFs and Rho GTPase-activating proteins (GAPs), and Vav2 has been shown to stimulate membrane ruffling and motility when overexpressed (Liu and Burridge, 2000;Marignani and Carpenter, 2001). Additional pathways leading to Rac1 activation also involve paxillin, as paxillin has been shown to participate in the targeting of active Rac1 to the membrane via an interaction with PKL, an ARF-GAP (Turner et al, 1999;Kondo et al, 2000). Thus, Nek3 contributes to the PRL-mediated phosphorylation of both Vav2 and paxillin and may regulate Rac1 activity through pathways involving both proteins, in a manner that will require additional study.…”

Paxillin: Adapting to Change

“…Paxillin can also interact with talin (223), poly(A)-binding protein 1 (307), ASAP1/2 (128,195), the nerve growth factor (NGF) receptor TrkA (178), the antiapoptosis protein bcl-2 (254,255), the focal adhesion protein TES (69), RACK (42), the serine/threonine phosphatase PP2A (110), the tyrosine phosphatase PTP- (206), Crk and CrkL (19,225,232), the tyrosine kinases Abl (143) and p210BCR/ABL (225), the protooncogene Cbl (224), and LIM kinase (62). Paxillin and Hic-5 also bind to the syndecan binding protein syndesmos (52), Hsp27 (118), Hsp72 (172), and the ring-finger ubiquitination component Rnf5 (54).…”

“…Importantly, Arf1-dependent paxillin shuttling between the Golgi and focal adhesions has been described (191), with the ArfGAPs, GIT2short, and ASAP2/PAP␣ implicated in this process (128,175). Other critical mediators of Golgi reorientation/polarization are Src (120) and p120RasGAP (135), which are paxillin binding partners (112,302).…”

TheDrosophilaARF6-GEF Schizo controls commissure formation by regulating Slit

“…(Someya et al, 2001) and promotes GDP/GTP exchange on ARF6. The small GTPase ARF6 is a plasma membrane-localized protein and functions in the regulation of membrane ruffling, cell motility, aspects of endocytosis and exocytosis, membrane recycling, reorganization of the cortical actin cytoskeleton and activation of phospholipase D (Kondo et al, 2000;Radhakrishna et al, 1999;Randazzo et al, 2000;Turner and Brown, 2001). In Drosophila Arf6 is remarkably well conserved, being more than 96% identical to the human counterpart (not shown).…”