Individual Cas phosphorylation sites are dispensable for processive phosphorylation by Src and anchorage-independent cell growth.
Cas is a multidomain signaling protein that resides in focal adhesions. Cas possesses a large central substrate domain containing 15 repeats of the sequence YXXP, which are phosphorylated by Src. The phosphorylation sites are essential for the roles of Cas in cell migration and in regulation of the actin cytoskeleton. We showed previously that Src catalyzes the multisite phosphorylation of Cas via a processive mechanism. In this study, we created mutant forms of Cas to identify the determinants for processive phosphorylation. Mutants containing single or multiple YXXP mutations were phosphorylated processively by Src, suggesting that individual sites are dispensable. The results also suggest that there is no defined order to the Cas phosphorylation events. We also studied the effects of these mutations by reintroducing Cas into Cas-deficient fibroblasts. Mutants lacking some or all YXXP sites augment the ability of Src to promote anchorage-independent growth. On the other hand, deletion of YXXP sites compromises the ability of Cas to promote tumor cell migration.
Patwardhan
Parag
P
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794.
Shen
Yongquan
Y
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084.
Goldberg
Gary S
GS
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084.
Miller
W Todd
WT
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794. Electronic address: todd.miller@stonybrook.edu.
D000818:Animals; D001665:Binding Sites; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein / Q000737:chemistry*; D005347:Fibroblasts / Q000378:metabolism; D007313:Insecta; D007700:Kinetics; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism; D010766:Phosphorylation; D019061:src-Family Kinases / Q000737:chemistry* / Q000378:metabolism
C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D019061:src-Family Kinases