Identification of a New Pyk2 Target Protein with Arf-GAP Activity

Andreev, Julian; Simon, Jean‐Pierre; Sabatini, D D; Kam, J; Plowman, Gregory D.; Randazzo, Paul A.; Schlessinger, Joseph

doi:10.1128/mcb.19.3.2338

Cited by 164 publications

(150 citation statements)

References 64 publications

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“…Neither ArfGAP activity nor the FAK-binding SHD1 domain was essential for PKL phosphorylation ( Figure 5). The precise role for PKL Arf-GAP activity in regulating cell protrusiveness, spreading and motility is unclear, although it is intriguing that FAK-and Src-family kinases interact with and phosphorylate a number of Arf-GAP proteins including ASAP1/2, ARAP3 and GIT1/GIT2 (Brown et al, 1998b;Andreev et al, 1999;Bagrodia et al, 1999;Zhao et al, 2000b;Haendeler et al, 2003;Randazzo and Hirsch, 2004;Stacey et al, 2004;Van Nieuw Amerongen et al, 2004;Yin et al, 2004). Additional work will be required to determine the precise role for PKL phosphorylation in generating a focal adhesion competent molecule.…”

Section: Discussionmentioning

confidence: 99%

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Brown

Cary

Jamieson

et al. 2005

MBoC

166

179

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The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility. INTRODUCTIONCell attachment, spreading, and motility are complex processes requiring the integration of diverse signaling networks and structural assemblies (Jockusch et al., 1995;Sastry and Burridge, 2000;Juliano, 2002). One of the earliest steps in transducing extracellular cues through integrins to the cytoskeleton is the activation of the tyrosine kinases Src and FAK (Schwartz et al., 1995;Giancotti and Tarone, 2003). FAK is an integrin-binding nonreceptor tyrosine kinase that upon integrin ligation is activated to autophosphorylate Y397 and bind to the Src SH2 domain (Schaller et al., 1994;Calalb et al., 1995). Src then phosphorylates FAK on multiple residues that increase FAK kinase activity and several downstream binding partners for Src/FAK are targeted for phosphorylation, including the focal adhesion proteins p130Cas and paxillin (reviewed in Brown and Turner, 2004;Playford and Schaller, 2004;Mitra et al., 2005). Phosphorylated paxillin binding to the SH2/SH3 adaptor protein Crk is implicated in Rac activation and stimulation of cell motility (Petit et al., 2000;Lamorte et al., 2003;Valles et al., 2004), whereas binding of paxillin to the p120RasGAP SH2 domain may displace and allow for the activation of p190RhoGAP and subsequent decrease in RhoA activity (Iwasaki et al., 2002).The Cdc42, Rac1, and RhoA members of the Ras superfamily of p21 GTPases are central intermediaries in coordinating the defined temporal-spatial progression of signals emanating from initial integrin ligation, through acquisition of a polarized state, to initiation and maintenance of directed ce...

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Section: Discussionmentioning

confidence: 99%

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Brown

Cary

Jamieson

et al. 2005

MBoC

166

179

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“…In addition, another ARF-GAP called PAG3 (paxillin-binding proteins bearing ARF-GAP motifs) also binds to paxillin, although the details of the interaction have not been fully elucidated (Kondo et al, 2000). Interestingly, PAG3 was also identi®ed as a CAKb/Pyk2-associated ARF-GAP called PAPa (Andreev et al, 1999).…”

Section: Paxillin Associated Enzymesmentioning

confidence: 99%

Paxillin: a focal adhesion-associated adaptor protein

2001

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Paxillin is a focal adhesion-associated, phosphotyrosinecontaining protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein ± protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/ threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a speci®c cellular compartment, the focal adhesions, and/ or to recruit speci®c combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility. Oncogene (2001) 20, 6459 ± 6472.

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“…Src and related tyrosine kinases have been implicated in regulating cell movement (29)(30)(31)(32). In addition, ASAP1 contains a C-terminal SH3 domain that binds to the focal adhesion tyrosine kinase FAK (J. T. Parsons, personal communication), whereas the ASAP1 relative PAP binds the FAK relative Pyk2 (28). ASAP1 and PAP also contain ankyrin repeats, a proline-rich region, and a pleckstrin homology (PH) domain.…”

mentioning

confidence: 99%

“…Two known Arf GAPs, ASAP1 (also called centaurin ␤4) and PAP (centaurin ␤3), bind known regulators of cytoskeleton organization, including phosphoinositides and Src family and focal adhesion kinase (FAK) family proteins (27,28). Furthermore, when transiently expressed, ASAP1 and PAP are found at the edge of transfected cells, where they could influence cortical actin during cell movement (27,28). ASAP1 was identified both as an Arf GAP, specific for Arf1 and Arf5 but with detectable activity on Arf6, and as a Src SH3-binding protein (27).…”

mentioning

confidence: 99%

The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton

Randazzo

Andrade

Miura

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

203

182

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Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin ␤ family, ASAP1 (also known as centaurin ␤4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.

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Identification of a New Pyk2 Target Protein with Arf-GAP Activity

Cited by 164 publications

References 64 publications

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Paxillin: a focal adhesion-associated adaptor protein

The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton

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