Characterization of an activated mutant of focal adhesion kinase: ‘SuperFAK’

Gabarra-Niecko, Veronica; Keely, Patricia J.; Schaller, Michael D.

doi:10.1042/bj20020065

Cited by 55 publications

(56 citation statements)

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“…A modest amount of PKL phosphorylation was detectable upon expression of kinase dead FAK, possibly due to transphosphorylation of Y397 by endogenous PYK2 (Li et al, 1999) allowing for some Src recruitment to KD FAK, or perhaps Src itself (Calalb et al, 1995). Constitutively active FAK, also called SuperFAK (Gabarra-Niecko et al, 2002), whose activity is independent of Src, efficiently restored PKL phosphorylation ( Figure 6B). This was similar to constitutively active Src (Y527F) rescuing the SYF null PKL phosphorylation defect ( Figure 6A).…”

Section: Pkl Tyrosine Phosphorylation Is Required For Paxillin Bindingmentioning

confidence: 92%

Section: Plasmids and Antibodiesmentioning

confidence: 99%

“…cDNAs encoding WT Csk and p130Cas cDNAs were provided by Akira Imamoto (University of Chicago, Chicago, IL), WT Abl by Jean Wang (University of California, San Diego, CA), WT Nck from Bruce Mayer (University of Connecticut, Storrs, CT), WT PTP-PEST from Michel Tremblay (McGill University, Montreal, Quebec, Canada). SuperFAK K578/581E (Gabarra-Niecko et al, 2002) and PKL mutants were generated by QuikChange mutagenesis (Stratagene, La Jolla, CA) and sequenced in their entirety at the SUNY Upstate DNA Core Facility (Syracuse, NY).Anti-PKL and anti-Hic-5 monoclonal antibodies were provided by BD Transduction Laboratories (Lexington, KY), anti-hemagglutinin (HA) monoclonal clone 12CA5 was obtained from Covance (Berkeley, CA), anti-Nck and anti-phosphotyrosine 4G10 were from Upstate Biotechnology (Charlottesville, VA), IgG-purified rabbit anti-green fluorescent protein (GFP) (Molecular Probes), and anti-T7 monoclonal was from Affinity Bioreagents (Golden, CO). …”

mentioning

confidence: 99%

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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

167

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: Pkl Tyrosine Phosphorylation Is Required For Paxillin Bindingmentioning

confidence: 92%

Section: Plasmids and Antibodiesmentioning

confidence: 99%

mentioning

confidence: 99%

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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

167

179

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“…Once phosphorylated on Tyr-925, FAK serves as a docking site for p85 regulatory subunit of PI3K (35-38) through recruitment of growth factor receptor-bound protein 2 (Grb2) (36), which is known to be involved in Raf/MEK/ERK signaling (35,36). We then focused on the activation of PI3K/AKT and MAPK/ERK in response to exogenous addition of PN at various time points.…”

Section: Pn Promotes ␤3-integrin/fak/akt/erk1/2 Activation In Chick Amentioning

confidence: 99%

Periostin Induces Intracellular Cross-talk between Kinases and Hyaluronan in Atrioventricular Valvulogenesis

Ghatak

Misra

Norris

et al. 2014

Journal of Biological Chemistry

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Background: Periostin-null mice exhibit a myxomatous atrioventricular valve phenotype. We propose two mechanisms as follows: periostin binds to collagen and links it to cell-surface receptors; periostin/␤-INTEGRIN signaling promotes valve morphogenesis. Results: Periostin/␤-INTEGRINs/focal adhesion kinase/PI3K/ERK signals promote hyaluronan synthase-2 activation, matrix remodeling, and valve progenitor cell survival/differentiation. Conclusion:The phenotype of periostin-null valves is consistent with a role for PN cell signaling through INTEGRIN receptors. Significance: Periostin is a valvulogenic signaling morphogen.

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“…T47D/tva cells, a derivative of the T47D breast cancer cell line that is susceptible to infection with avian retroviral vectors, were used for this analysis (13). T47D/tva cells were infected with the empty RCAS A vector or with RCAS A containing the wild-type FAK or KAKTLR cDNA.…”

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confidence: 99%

FERM Domain Interaction Promotes FAK Signaling

Dunty¹,

Gabarra-Niecko²,

King³

et al. 2004

Molecular and Cellular Biology

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113

116

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From the results of deletion analyses, the FERM domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM domain.The focal adhesion kinase, FAK, plays a major role in transducing signals downstream of integrins (40,47). Upon integrinmediated adhesion, FAK becomes tyrosine phosphorylated and activated. Additional signaling molecules, e.g., Src and phosphatidylinositol 3-kinase, are recruited into complexes with FAK, leading to the transduction of biochemical signals that control important biological processes. Integrin signaling via FAK regulates cell migration, proliferation, and survival. FAK-dependent regulation of one or more of these processes is essential, since fak Ϫ/Ϫ mice exhibit embryonic lethality (27). Conversely, enhanced FAK signaling may lead to aberrant cell proliferation, survival, or migration, which may have pathological consequences in humans. For example, aberrant FAK signaling may contribute to cancer development and progression to metastatic disease (40).FAK contains three major domains, an N-terminal domain, a central catalytic domain, and a C-terminal domain (40, 47). The C-terminal domain can be further subdivided into the focal adhesion targeting (FAT) sequence, comprising the Cterminal 140 amino acids of the protein, and the region between the catalytic domain and the FAT sequence. Focal adhesion-associated FAT sequence binding partners have been identified, and insight into the molecular basis of FAT sequence function was recently obtained from crystal and nuclear magnetic resonance structure analyses (3,14,23,26,31). The sequence between the catalytic domain and the FAT sequence contains docking sites for SH3 domain-containing proteins and thus serves as a scaffold for the recruitment of signaling proteins. Several sites of tyrosine phosphorylation play important regulatory roles in FAK. Within the catalytic domain, two tyrosine residues in the activation loop, tyrosines 576 and 577, regulate catalytic activity. The major site of autophosphorylation, tyrosine 397, lies just N terminal to the catalytic domain and serves as a binding site for Src family kinases. While details regarding the function of the catalytic and C-terminal domains have been elucidated, fewer studies have examined the function of ...

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Characterization of an activated mutant of focal adhesion kinase: ‘SuperFAK’

Cited by 55 publications

References 56 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

Periostin Induces Intracellular Cross-talk between Kinases and Hyaluronan in Atrioventricular Valvulogenesis

FERM Domain Interaction Promotes FAK Signaling

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