Functional analysis of Src homology 3-encoding exon (exon 2) of p130Cas in primary fibroblasts derived from exon 2-specific knockout mice.
p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of a Src homology 3 (SH3) domain, a substrate domain (SD) and a Src binding domain (SBD). The SH3 domain of Cas associates with focal adhesion kinase (FAK), but its role in cellular function has not fully been understood. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking exon 2, which encodes the SH3 domain (Cas Deltaexon 2). In comparison to wild-type cells, Cas exon 2(Delta/Delta) cells showed reduced motility, which could be due to impaired tyrosine-phosphorylation of FAK and Cas, reduced FAK/Cas/Src/CrkII binding, and also impaired localization of Cas Deltaexon 2 to focal adhesions on fibronectin. In addition, to analyze downstream signaling pathways regulated by Cas exon 2, we performed microarray analyses. Interestingly, we found that a deficiency of Cas exon 2 up-regulated expression of CXC Chemokine Receptor-4 and CC Chemokine Receptor-5, which may be regulated by IkappaBalpha phosphorylation. These results indicate that the SH3-encoding exon of Cas participates in cell motility, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation, recruitment of Cas to focal adhesions and regulation of cell motility-associated gene expression in primary fibroblasts.
Tazaki
Tatsuya
T
Department of Developmental Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
Miyazaki
Kazuko
K
Hiyama
Eiso
E
Nakamoto
Tetsuya
T
Sakai
Ryuichi
R
Yamasaki
Norimasa
N
Honda
Zen-Ichiro
Z
Noda
Masaki
M
Miyasaka
Nobuyuki
N
Sueda
Taijiro
T
Honda
Hiroaki
H
D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000737:chemistry / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D015870:Gene Expression; D020672:I-kappa B Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D046912:Multiprotein Complexes; D000072000:NF-KappaB Inhibitor alpha; D050737:Proto-Oncogene Proteins c-crk / Q000737:chemistry / Q000378:metabolism; D019713:Receptors, CCR5 / Q000235:genetics; D019718:Receptors, CXCR4 / Q000235:genetics; D017384:Sequence Deletion; D015398:Signal Transduction; D019699:Thrombospondins / Q000235:genetics; D014945:Wound Healing; D018909:src Homology Domains
C493842:Bcar1 protein, mouse; C514936:CXCR4 protein, mouse; D050719:Crk-Associated Substrate Protein; D020672:I-kappa B Proteins; D046912:Multiprotein Complexes; C000606205:Nfkbia protein, mouse; D050737:Proto-Oncogene Proteins c-crk; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D019699:Thrombospondins; C101864:thrombospondin 4; D000072000:NF-KappaB Inhibitor alpha; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse