scholarly journals The Association of ASAP1, an ADP Ribosylation Factor-GTPase Activating Protein, with Focal Adhesion Kinase Contributes to the Process of Focal Adhesion Assembly

2002 ◽  
Vol 13 (6) ◽  
pp. 2147-2156 ◽  
Author(s):  
Yunhao Liu ◽  
Joost C. Loijens ◽  
Karen H. Martin ◽  
Andrei V. Karginov ◽  
J. Thomas Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Ph Domain ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Transient Overexpression ◽  
Adp Ribosylation Factor

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src. Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). GlutathioneS-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.

scholarly journals The Cytoplasmic Tyrosines of Integrin Subunit β1 Are Involved in Focal Adhesion Kinase Activation

2000 ◽  
Vol 20 (15) ◽  
pp. 5758-5765 ◽  
Author(s):  
Krister Wennerberg ◽  
Annika Armulik ◽  
Takao Sakai ◽  
Marjam Karlsson ◽  
Reinhard Fässler ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Phosphorylation Site ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Wild Type ◽  
Directed Cell Migration ◽  
Dependent Cell

ABSTRACT We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit β1 (Y783 and Y795) to phenylalanines markedly reduces the capability of β1A integrins to mediate directed cell migration. In this study, β1-dependent cell spreading was found to be delayed in GD25 cells expressing β1AY783/795F compared to that in wild-type GD25-β1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to β1-dependent adhesion in GD25-β1AY783/795F cells compared to that in wild-type GD25-β1A or mutants in which only a single tyrosine was altered (β1AY783F or β1AY795F). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via β1AY783/795F lies at the level of the initial autophosphorylation step. Indeed, β1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the β1AY783/795F cells, consistent with the impairment in FAK activation. In contrast, p130CAS overall tyrosine phosphorylation was unaffected by the β1 mutations. Despite the defect in β1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-β1AY783/795F cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit β1A are critical mediators of FAK activation and cell spreading in GD25 cells.

Concerted modulation of paxillin dynamics at focal adhesions by deleted in liver cancer-1 and focal adhesion kinase during early cell spreading

Cytoskeleton ◽  
2014 ◽  
Vol 71 (12) ◽  
pp. 677-694 ◽  
Author(s):  
Shelly Kaushik ◽  
Archna Ravi ◽  
Feroz M. Hameed ◽  
Boon Chuan Low
Keyword(s):  
Liver Cancer ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading

scholarly journals Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A

1997 ◽  
Vol 324 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Alan RICHARDSON ◽  
John D. SHANNON ◽  
Reid B. ADAMS ◽  
Michael D. SCHALLER ◽  
J. Thomas PARSONS
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Serine Phosphorylation ◽  
Early Event ◽  
Kinase A

Focal adhesion kinase (pp125FAK) is a protein tyrosine kinase that is localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matrix. In some cells the C-terminal non-catalytic domain of pp125FAK is expressed as a separate protein referred to as FRNK (FAK-related, non-kinase). We have previously shown that overexpression of FRNK inhibits tyrosine phosphorylation of pp125FAK and its substrates as well as inhibiting cell spreading on fibronectin. In this report we identify Ser148 and Ser151 as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125FAK and in response to integrin binding to fibronectin. Tyrosine phosphorylation of pp125FAK appears to be an early event after integrin occupancy, and serine phosphorylation of FRNK occurs significantly later. Treatment of fibroblasts with a series of protein kinase A inhibitors delayed serine phosphorylation of FRNK as well as cell spreading on fibronectin and tyrosine phosphorylation of pp125FAK. However, these PKA inhibitors are unlikely to delay cell spreading simply by preventing serine phosphorylation of FRNK, as overexpression of FRNK containing mutations of Ser148 and Ser151 either singly or jointly to either alanine or glutamate residues did not significantly alter the ability of FRNK to act as an inhibitor of pp125FAK.

scholarly journals 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

2000 ◽  
Vol 11 (4) ◽  
pp. 1315-1327 ◽  
Author(s):  
Akiko Kondo ◽  
Shigeru Hashimoto ◽  
Hajime Yano ◽  
Kuniaki Nagayama ◽  
Yuichi Mazaki ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Binding Protein ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Gtpase Activating Protein ◽  
Protein Activity ◽  
Adp Ribosylation ◽  
Focal Contacts ◽  
Adp Ribosylation Factor

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/Papα, 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.

scholarly journals Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate

2019 ◽  
Vol 294 (46) ◽  
pp. 17354-17370 ◽  
Author(s):  
Neeladri Sekhar Roy ◽  
Xiaoying Jian ◽  
Olivier Soubias ◽  
Peng Zhai ◽  
Jessica R. Hall ◽  
...  
Keyword(s):  
Point Mutations ◽  
Ph Domain ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Src Homology 3 ◽  
N Terminus ◽  
Src Homology ◽  
Mechanistic Basis ◽  
Α Helix ◽  
Adp Ribosylation Factor

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus–containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2–17 of ARF1 ([2–17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first β-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2–17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.

scholarly journals Arrestins regulate cell spreading and motility via focal adhesion dynamics

2015 ◽  
Vol 26 (4) ◽  
pp. 622-635 ◽  
Author(s):  
Whitney M. Cleghorn ◽  
Kevin M. Branch ◽  
Seunghyi Kook ◽  
Christopher Arnette ◽  
Nada Bulus ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Wild Type

Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin. Cells lacking both nonvisual arrestins showed excessive spreading on fibronectin and poly-d-lysine, increased adhesion, and reduced motility. The absence of arrestins greatly increases the size and lifespan of FAs, indicating that arrestins are necessary for rapid FA turnover. In nocodazole washout assays, FAs in arrestin-deficient cells were unresponsive to disassociation or regrowth of microtubules, suggesting that arrestins are necessary for microtubule targeting–dependent FA disassembly. Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells. In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype. Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

scholarly journals Src SH2 Arginine 175 Is Required for Cell Motility: Specific Focal Adhesion Kinase Targeting and Focal Adhesion Assembly Function

2006 ◽  
Vol 26 (12) ◽  
pp. 4399-4409 ◽  
Author(s):  
Myeong Gu Yeo ◽  
Michael A. Partridge ◽  
Ellen J. Ezratty ◽  
Qiong Shen ◽  
Gregg G. Gundersen ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Src Kinase ◽  
Focal Adhesions ◽  
Sh2 Domain ◽  
Wild Type ◽  
Binding Function ◽  
Generation Capacity ◽  
Substrate Phosphorylation

ABSTRACT Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.

scholarly journals Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: Implications in cancer cell biology

2006 ◽  
Vol 97 (9) ◽  
pp. 801-806 ◽  
Author(s):  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Waka Natsume ◽  
Tetsuo Torii ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor
Sign in / Sign up

Export Citation Format

Share Document