CrkL directs ASAP1 to peripheral focal adhesions.
Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.
Oda
Atsushi
A
Laboratory of Environmental Biology, Department of Preventive Medicine, Hokkaido University School of Medicine, Sapporo 060-8638, Japan. aoda@med.hokudai.ac.jp
Wada
Ikuo
I
Miura
Koichi
K
Okawa
Katsuya
K
Kadoya
Toshihiko
T
Kato
Takashi
T
Nishihara
Hiroshi
H
Maeda
Masae
M
Tanaka
Shinya
S
Nagashima
Kazuo
K
Nishitani
Chiaki
C
Matsuno
Kazuhiko
K
Ishino
Masaho
M
Machesky
Laura M
LM
Fujita
Hiroyoshi
H
Randazzo
Paul
P
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000097:blood*; D002448:Cell Adhesion / Q000502:physiology*; D002522:Chlorocebus aethiops; D022001:Focal Adhesions / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000097:blood* / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010973:Platelet Adhesiveness / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D002352:Carrier Proteins; D008024:Ligands; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins