Requirements for localization of p130cas to focal adhesions.
p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.
Nakamoto
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Japan.
Sakai
R
R
Honda
H
H
Ogawa
S
S
Ueno
H
H
Suzuki
T
T
Aizawa
S
S
Yazaki
Y
Y
Hirai
H
H
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002448:Cell Adhesion; D002451:Cell Compartmentation; D002471:Cell Transformation, Neoplastic / Q000473:pathology*; D050719:Crk-Associated Substrate Protein; D019084:Fluorescent Antibody Technique, Indirect; D016391:Genes, src; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130
C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130; D016392:Proto-Oncogene Proteins pp60(c-src)