Protein Tyrosine Phosphatase α Phosphotyrosyl-789 Binds BCAR3 To Position Cas for Activation at Integrin-Mediated Focal Adhesions

Abstract

Integrin-mediated focal adhesions connect the extracellular matrix and cytoskeleton to regulate cell responses, such as migration. Protein tyrosine phosphatase ␣ (PTP␣) regulates integrin signaling, focal adhesion formation, and migration, but its roles in these events are incompletely understood. The integrin-proximal action of PTP␣ activates Src family kinases, and subsequent phosphorylation of PTP␣ at Tyr789 acts in an unknown manner to promote migration. PTP␣-null cells were used in reconstitution assays to distinguish PTP␣-Tyr789-dependent signaling events. This showed that PTP␣-Tyr789 regulates the localization of PTP␣ and the scaffolding protein Cas to adhesion sites where Cas interacts with and is phosphorylated by Src to initiate Cas signaling. Linking these events, we identify BCAR3 as a molecular connector of PTP␣ and Cas, with phospho-Tyr789 PTP␣ serving as the first defined cellular ligand for the BCAR3 SH2 domain that recruits BCAR3-Cas to adhesions. Our findings reveal a novel role of PTP␣ in integrin-induced adhesion assembly that enables Src-mediated activation of the pivotal function of Cas in migration.

Association of the Breast Cancer Antiestrogen Resistance Protein 1 (BCAR1) and BCAR3 Scaffolding Proteins in Cell Signaling and Antiestrogen Resistance

“…Overall, these results suggest that the antiestrogen resistance conferred by BCAR3 depends on its ability to stabilize BCAR1 as well as activate SRC and promote BCAR1 tyrosine phosphorylation, leading to downstream signaling (9,11,37). Accordingly, SRC activation was most elevated in the three cell populations expressing wild-type BCAR3, the same ones that grew best in the presence of ICI182780.…”

“…In contrast, the NSP3␤ isoform contains a shorter N-terminal region of ϳ60 amino acids and, like BCAR3, is not constitutively localized at the plasma membrane. However, both NSP3␤ and BCAR3 can localize at the membrane through binding of their SH2 domains to tyrosine-phosphorylated motifs of membrane-associated proteins, which in turn leads to increased SRC activity and BCAR1 phosphorylation (11,25,31,32,35,37,40,50).…”

“…However, the precise role of BCAR1 serine phosphorylation (which does not seem to be important for antiestrogen resistance) and the kinase(s) responsible remain to be identified (39). On the other hand, SRC activation by BCAR3 (30,32) has been recently shown to promote phosphorylation of the Tyr-789 motif of PTP␣, a receptor-type phosphatase that can potentiate SRC activation by dephosphorylating its inhibitory Tyr-527 phosphorylation site (37). Binding of the BCAR3 SH2 domain to phosphorylated PTP␣ leads to recruitment of the associated BCAR1, positioning it for SRC-mediated phosphorylation (32,37).…”

“…Thus, the R743A mutation has been reported to affect some BCAR3 functions. For example, it can inhibit BCAR3 association with the SRC kinase and BCAR3-dependent SRC activation as well as impair BCAR3-dependent fibroblast migration (37,39,51).…”

“…This distinctive structure includes an N-terminal segment subject to alternative splicing, a SH2 domain that can bind activated receptor-tyrosine kinases and the PTP␣ phosphatase (at least in the case of BCAR3), and a C-terminal CDC25-type guanine nucleotide exchange factor (GEF)-like domain (11,36,37). Recent x-ray crystallography studies have shown that this GEF-like domain has a unique conformation that is incompatible with the binding of RAS GTPases but is capable of functioning as an adaptor module that mediates extremely tight binding to the C-terminal focal adhesion targeting domain of CAS family proteins.…”