Direct binding of C-terminal region of p130Cas to SH2 and SH3 domains of Src kinase.
p130Cas is a major tyrosine-phosphorylated protein that tightly binds v-Crk in v-crk-transformed cells and v-Src in v-src-transformed cells. The "substrate domain" of p130Cas contains 15 possible Src homology (SH) 2-binding motifs, most of which conform to the binding motif for the Crk SH2 domain. Another region near its C terminus contains possible binding motifs for the Src SH2 domain and proline-rich sequences that are candidates for SH3-binding sites. Using GST fusion proteins, we revealed that both SH2 and SH3 domains of Src bind p130Cas, whereas v-Crk binds p130Cas through its SH2 domain. We located the binding site of p130Cas for the Src SH3 domain at the sequence RPLPSPP in the region near its C terminus. Mutations within this sequence or at Tyr762 of p130Cas caused a significant reduction in the association of p130Cas with Src, and no association was detected when both of them were deleted. The kinase activity in v-Crk-transformed cells was also associated with p130Cas through this region. On the other hand, the deletion of the substrate domain abolished the binding with v-Crk. The association through the C-terminal region of p130Cas with Src kinase may facilitate effective hyperphosphorylation of tyrosine residues in the substrate domain of p130Cas, resulting in the binding of SH2-containing molecules to p130Cas.
Nakamoto
T
T
Molecular Biology Division, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi-machi, Kawachi-gun, Tochigi, 329-04 Japan.
Sakai
R
R
Ozawa
K
K
Yazaki
Y
Y
Hirai
H
H
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D050719:Crk-Associated Substrate Protein; D051379:Mice; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D018909:src Homology Domains
C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015682:Retroviridae Proteins, Oncogenic; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)