Subsets of the major tyrosine phosphorylation sites in Crk-associated substrate (CAS) are sufficient to promote cell migration.
Crk-associated substrate (p130(CAS) or CAS) is a major integrin-associated Src substrate that undergoes tyrosine phosphorylation at multiple YXXP motifs in its substrate domain (SD) to create docking sites for SH2-containing signaling effectors. Notably, recruitment of Crk adaptor proteins to the CAS SD sites is implicated in promoting cell migration. However, it is unclear which or how many of the 15 CAS SD YXXP tyrosines are critically involved. To gain a better understanding of CAS SD function, we assessed the signaling capacity of individual YXXP motifs. Using site-directed mutagenesis combined with tryptic phosphopeptide mapping, we determined that the ten tyrosines in YXXP motifs 6-15 are the major sites of CAS SD phosphorylation by Src. Phosphopeptide binding assays showed that all of these sites are capable of binding the Crk SH2 domain. To evaluate the requirement for CAS YXXP sites in stimulating cell migration, a series of phenylalanine substitution variants were expressed in CAS -/- mouse embryo fibroblasts. CAS expression enhanced the rate of cell migration into a monolayer wound in a manner dependent on the major sites of Src phosphorylation. Effective wound healing was achieved by CAS variants containing as few as four of the major sites, indicating sufficiency of partial SD signaling function in this cell migration response.
Shin
Nah-Young
NY
Department of Cell and Developmental Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Dise
Rebecca S
RS
Schneider-Mergener
Jens
J
Ritchie
Marylyn D
MD
Kilkenny
Dawn M
DM
Hanks
Steven K
SK
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*; D014945:Wound Healing; D018909:src Homology Domains
C493841:BCAR1 protein, human; C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D010455:Peptides; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D010649:Phenylalanine; D014357:Trypsin