The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly.
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 pp60(Src). 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). Glutathione S-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.
Liu
Yunhao
Y
Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia 22908, USA.
Loijens
Joost C
JC
Martin
Karen H
KH
Karginov
Andrei V
AV
Parsons
J Thomas
JT
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000502:physiology*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016335:Zinc Fingers
D002352:Carrier Proteins; D017931:DNA Primers; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse