Breast cancer anti-estrogen resistance 3 (BCAR3) protein augments binding of the c-Src SH3 domain to Crk-associated substrate (p130cas).
The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.
Department of Medicine, Section of Hematology/Oncology, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts 02118, USA.
Anthony
A
Makkinje
Pierre
P
Vanden Borre
Richard I
RI
Near
Prayag S
PS
Patel
Adam
A
Lerner
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D050719:Crk-Associated Substrate Protein / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C493841:BCAR1 protein, human; C118588:BCAR3 protein, human; D050719:Crk-Associated Substrate Protein; D020662:Guanine Nucleotide Exchange Factors; D046912:Multiprotein Complexes; D019061:src-Family Kinases