Chat, a Cas/HEF1-associated Adaptor Protein That Integrates Multiple Signaling Pathways

Seisuke Hattori,Akira Sakakibara

doi:10.1074/jbc.275.9.6404

Seisuke Hattori, Akira Sakakibara

Open Access

https://doi.org/10.1074/jbc.275.9.6404

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Abstract

Cas (Crk-associated substrate) and HEF1 (human enhancer of filamentation) are related adaptor proteins that function in integrin-mediated cell adhesion and antigen receptor signaling pathways. We report here a molecular cloning of Chat (Cas/HEF1-associated signal transducer) that associates with Cas and HEF1. Chat is a 78-kDa signaling molecule with an N-terminal SH2 domain and is expressed in a wide range of tissues. In hematopoietic cells, a 115-kDa isoform of Chat (Chat-H) was specifically expressed. Chat is associated with Cas in brain, and Chat-H is associated with HEF1 in splenocytes. Deletion analyses revealed that Chat and Cas are associated with each other by their C-terminal domains. Treatment of PC12 cells with epidermal growth factor or nerve growth factor increased the phosphorylation level of Chat. This increase was suppressed by an inhibitor of mitogen-activated protein (MAP) kinase kinase, PD98059, suggesting the phosphorylation of Chat by MAP kinase. In Chat-overexpressed COS7 cells, the activity of c-Jun N-terminal kinase was up-regulated. After the epidermal growth factor stimulation, Chat and Cas were colocalized with actin filaments at ruffling membranes. These findings suggest that Chat transduces signals of tyrosine kinases and MAP kinase to Cas signaling pathway.

Highlights

Cell adhesion to extracellular matrix (ECM)1 concerns various biological events, such as cell growth, survival, differentiation, and migration, which is mediated by integrin receptors in a wide range of cell types
This study demonstrates that Chat, an adaptor protein having an Src homology 2 (SH2) domain, interacts with the C-terminal domain of Cas family adaptor proteins
Chat showed colocalization with Cas at ruffling membranes. These results suggest that Chat integrates signals from tyrosine kinases and mitogen-activated protein (MAP) kinase, controlling cell growth and cytoskeletal reorganization through the Cas signaling pathway

Summary

EXPERIMENTAL PROCEDURES

Materials and Cells—In the process of an immunoscreening of a mouse lung cDNA library constructed in ␭gt using a monoclonal antibody B45/18, the initial clone was isolated by a cross-reaction of the antibody. Using this clone as a probe, full-length mouse cDNA encoding Chat was obtained. After 24 h, cells were homogenized in His-binding buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol), and clarified by centrifugation at 15,000 ϫ g for 20 min. To express glutathione S-transferase (GST) fusion protein of Chat association domain of Cas (GST-Cas671–874) in E. coli, pGEX-4T-1 vector (Amersham Pharmacia Biotech) was used. GST or GST-Cas671874 produced in E. coli was bound to glutathione-Sepharose (Amersham Pharmacia Biotech) in Binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol). The specimens were incubated with first antibodies for 60 min in a moist chamber, washed three times with the blocking solution, and incubated for 60 min with the second antibody, Cy2-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-mouse IgG (Amersham Pharmacia Biotech). Samples were washed with PBS for three times, mounted in Perma Fluor (Immunon), and examined using a fluorescence microscope, Zeiss Axiophot II photomicroscope (Carl Zeiss)

RESULTS

DISCUSSION

Akira Sakakibara and Seisuke Hattori