SHEP1 Function in Cell Migration Is Impaired by a Single Amino Acid Mutation That Disrupts Association with the Scaffolding Protein Cas but Not with Ras GTPases

Abstract

SHEP1 is a signaling protein that contains a guanine nucleotide exchange factor-like domain, which binds Ras family GTPases and also forms a stable complex with the scaffolding protein Crk-associated substrate (Cas). SHEP1 and Cas have several common functions, such as increasing c-Jun N-terminal kinase activity, promoting T cell activation, and regulating the actin cytoskeleton. However, it is unclear whether a physical association between SHEP1 and Cas is required for these activities. We reported previously that SHEP1 is tyrosine-phosphorylated downstream of the EphB2 receptor; in this study, we further demonstrate that activated EphB2 inhibits SHEP1 association with Cas. To investigate whether phosphorylation negatively regulates the SHEP1-Cas complex, we have identified by mass spectrometry several SHEP1 tyrosine phosphorylation sites downstream of EphB2; of particular interest among them is tyrosine 635 in the Cas association/exchange factor domain. Mutation of this tyrosine to glutamic acid, but not to phenylalanine, disrupts Cas binding to SHEP1 without inhibiting Ras GTPase binding. The glutamic acid mutation also makes SHEP1 unable to promote Cas-Crk association, membrane ruffling, and cell migration toward epidermal growth factor (EGF), implying that these activities of SHEP1 depend upon a physical interaction with Cas. Association with Cas also seems to be necessary for EGF-induced SHEP1 tyrosine phosphorylation, which is mediated by a Src family kinase. It is noteworthy that EGF stimulation does not cause dissociation of SHEP1 from Cas. These data show that SHEP1 regulates membrane ruffling and cell migration and that binding to Cas is probably critical for these functions. Furthermore, the SHEP1-Cas complex may have different roles downstream of EphB2 and the EGF receptor.

SHEP1 partners with CasL to promote marginal zone B-cell maturation

“…Our observation that MZ B cells are absent in SHEP1-deficient mice prompted us to investigate the underlying mechanisms of this B cell-intrinsic defect. Because SHEP1 has been shown to regulate the migration of COS cells and T cells (21,28), we reasoned that SHEP1 might also regulate Bcell migration. Indeed, in the absence of SHEP1, B cells revealed impaired migration toward CXCL13 and S1P.…”

“…The SHEP1 gene consists of 15 exons with alternative splicing occurring among the first six exons, resulting in isoforms SHEP1α (long), SHEP1β, SHEP1γ, and SHEP1δ (19). The N-terminal SH2 domain is followed by a pro/ser rich domain and a C-terminal GEFlike domain, which has been shown to bind R-Ras, Rap1, and Rap2 (20,21).Using conventional and B cell-specific conditional SHEP1-deficient mice, we show that the loss of SHEP1 results in a marked reduction in mature MZ B cells, whereas the MZ B-cell precursor population remains intact. SHEP1-deficient B cells show defects in migration toward CXCL13 and S1P.…”

“…The SHEP1 gene consists of 15 exons with alternative splicing occurring among the first six exons, resulting in isoforms SHEP1α (long), SHEP1β, SHEP1γ, and SHEP1δ (19). The N-terminal SH2 domain is followed by a pro/ser rich domain and a C-terminal GEFlike domain, which has been shown to bind R-Ras, Rap1, and Rap2 (20,21).…”

Bidirectional ephrin/Eph signaling in synaptic functions

“…Through a direct interaction between the SH2-domain of SHEP1 and a conserved phosphotyrosine motif the juxtamembrane region, activated Eph receptors can be coupled to R-Ras and Rap1A [20]. Eph receptor activation also regulates membrane ruffling and cell migration by inhibiting the interaction between SHEP1 and Cas [14]. In addition to GEFs, Eph receptors can also associate with the p120-Ras GTPase activating protein (p120-RasGAP) upon ligand binding through SH2 mediated interactions [15,23,47].…”

“…Another actin modulating protein downstream of Eph forward signaling is SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which binds Ras family GTPases (i.e. R-Ras and Rap1A) and also forms a stable complex with the scaffolding protein Crk-associated substrate (Cas) [14,20]. Through a direct interaction between the SH2-domain of SHEP1 and a conserved phosphotyrosine motif the juxtamembrane region, activated Eph receptors can be coupled to R-Ras and Rap1A [20].…”