T Cell Receptor-mediated Tyrosine Phosphorylation of Cas-L, a 105-kDa Crk-associated Substrate-related Protein, and Its Association of Crk and C3G

Abstract

Cas-L (pp105), a Crk-associated substrate (p130Cas )-related protein, was first identified as a 105-kDa protein that is tyrosine-phosphorylated following ␤1 integrin cross-linking in T cells. Cas-L contains possible multiple binding sites for the Src homology (SH) 2 domains of various signaling molecules, and appears to be involved in signal transduction through phosphorylated tyrosine-mediated protein-protein interaction. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocytespecific signals. Here, we show the involvement of Cas-L in the T cell receptor (TCR)/CD3 signaling pathway. Cas-L is transiently phosphorylated following CD3 cross-linking, and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant that lacks the SH3 domain, the binding site for focal adhesion kinase (FAK), is also tyrosine-phosphorylated upon CD3 cross-linking, but not upon ␤1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Taken together, the present study indicates a novel signaling pathway mediated by tyrosine-phosphorylated Cas-L upon the TCR/CD3 stimulation. T cell receptor (TCR)1 -antigen binding induces gene expression, cytokine production, and cell proliferation in T lymphocytes (1). These TCR-dependent signals are mediated by tyrosine phosphorylation of various proteins including CD3␦, CD3⑀, CD3␥, Shc, Vav,. These signaling molecules appear to be involved in the phosphorylated tyrosinemediated protein-protein interaction and the recruitment of the other signaling molecules containing Src homology (SH) 2 domains (3). The recruitment of these signaling molecules is essential to induce various signals to the downstream events such as the activation of mitogen-activated protein kinases, Ca 2ϩ influx, and transcriptional activation of various genes (1).Thus, protein tyrosine phosphorylation plays a key role during the initial phase of TCR-mediated T cell activation. However, the essential phosphorylated molecule and the precise function of each phosphorylated molecule for these signaling pathwayshave not yet been clarified. Cas-L (pp105) was first identified as a 105-kDa protein that is tyrosine-phosphorylated upon ␤1 integrin cross-linking (4). Cas-L belongs to Cas-family along with p130Cas and Efs/Sin (5-7). The three proteins share the structural characteristics of an N-terminal SH3 domain, followed by multiple (8 to 15) YXXP motifs, which are putative binding sites for the Crk SH2 domain, and a conserved YDYVHL sequence that possibly binds to the Src SH2 domain. (5-7). Cas-L was shown to associate with the C-terminal domain of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that is localized to focal adhesions (5,8). Recently, we reported that the conserved YDYVHL sequence of Cas-L was phosphorylated by FAK following ␤1 integrin cross-linking and that an Src family tyrosine kinase is recruited to the phosphorylated YDYVHL sequence and causes further tyrosine phosphorylation of ...

Crk-associated substrate lymphocyte type regulates transforming growth factor-β signaling by inhibiting Smad6 and Smad7

“…A unique aspect of our data is that the observed Cas-L/ I-Smad interaction is not based on specific Cas-L domain, whereas other substrates for Cas-L, including FAK, Crk and Src, interact with its SH3 domain, substrate domain and YDYVHL motif, respectively (Tachibana et al, 1997;Ohashi et al, 1998). Additional supporting evidence is that only wt Cas-L, and not the mutants lacking the aforementioned domains, affects the sensitivity of Huh-7 cells to TGF-b.…”

“…Our work also indicates that Cas-L/I-Smads interaction influences the recruitment of I-Smads to TGF-b type I receptor. In the A member of the Cas family proteins, which include p130Cas, Efs/Sin and Cas-L/HEF1, Cas-L has a described physiological role at focal adhesion sites (Ohashi et al, 1998). Accumulating evidence strongly suggests that Cas-L is a multifunctional docking protein, with a role in cell motility (van Seventer et al, 2001;Seo et al, 2005), cytokine production , cell shape change , cell cycle (Astier et al, 1997) and cell division (Pugacheva and Golemis, 2005), besides cell adhesion.…”

“…We previously demonstrated that the 105 kDa protein (pp105) termed Cas-L (Cas lymphocyte type) (Minegishi et al, 1996) or human enhancer of filamentation 1 (HEF1) (Law et al, 1996) is a focal adhesion kinase (FAK)-associated docking protein that is heavily tyrosine phosphorylated by FAK and Src family kinases upon engagement with b1 integrin and the T-cell antigen receptor complex in T cells (Minegishi et al, 1996;Tachibana et al, 1997;Ohashi et al, 1998). Transfection of Cas-L into Jurkat cells markedly enhanced cell motility and interleukin-2 production following b1 integrin engagement.…”

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer

“…22,23 This hyperphosphorylation is a hallmark of the active signaling form of CAS proteins and generates binding sites for CRK adaptors, which then recruit the nucleotide exchange factors DOCK180 and C3G. [24][25][26][27] DOCK180 is a key activator of the RHO family GTPase RAC1, which in turn controls activation of the JNK MAP kinase and actin cytoskeleton remodeling, while C3G activates the RAS family GTPase RAP1, leading to increased integrin activity. 25,[28][29][30][31][32][33] Together, these 2 pathways promote cell substrate adhesion, migration, and invasiveness as well as proliferation.…”

Focal adhesion kinase in integrin-mediated signaling

“…The first was named both Efs (90) and Sin (91), while the second was named both HEF-1 (92) and CasL (93). Both homologs are structurally similar to p130…”