ABSTRACT
p130(cas) (Crk-associated substrate) is a docking protein that is involved in assembly of focal adhesions and concomitant cellular signaling. It plays a role in physiological regulation of cell adhesion, migration, survival, and proliferation, as well as in oncogenic transformation. The molecule consists of multiple protein-protein interaction motifs, including a serine-rich region that is positioned between Crk and Src-binding sites. This study reports the first structure of a functional domain of Cas. The solution structure of the serine-rich region has been determined by NMR spectroscopy, demonstrating that this is a stable domain that folds as a four-helix bundle, a protein-interaction motif. The serine-rich region bears strong structural similarity to four-helix bundles found in other adhesion components like focal adhesion kinase, alpha-catenin, or vinculin. Potential sites for phosphorylation and interaction with the 14-3-3 family of cellular regulators are identified in the domain and characterized by site-directed mutagenesis and binding assays. Mapping the degree of amino acid conservation onto the molecular surface reveals a patch of invariant residues near the C terminus of the bundle, which may represent a previously unidentified site for protein interaction.
Subject(s)
Proteins/physiology , Serine/chemistry , 14-3-3 Proteins/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cell Adhesion , Cell Movement , Cell Proliferation , Cell Survival , Cell Transformation, Neoplastic , Crk-Associated Substrate Protein , Cytoskeletal Proteins/chemistry , Humans , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Proteins/chemistry , Rats , Retinoblastoma-Like Protein p130 , Sequence Homology, Amino Acid , Signal Transduction , Vinculin/chemistry , alpha CateninABSTRACT
The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.
Subject(s)
Proteins/chemistry , 14-3-3 Proteins/chemistry , Animals , Cell Transformation, Neoplastic , Circular Dichroism , Crk-Associated Substrate Protein , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Integrins , Light , Magnetic Resonance Spectroscopy , Phosphorylation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 12 , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Proteins/metabolism , Rats , Retinoblastoma-Like Protein p130 , Scattering, Radiation , Serine/chemistry , Signal Transduction , src Homology Domains , src-Family Kinases/metabolismABSTRACT
Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.
Subject(s)
Adaptor Proteins, Signal Transducing , CD40 Antigens/biosynthesis , Receptors, Tumor Necrosis Factor/chemistry , Signal Transduction , Amino Acid Motifs , Amino Acid Sequence , CD40 Antigens/chemistry , Cell Line , Cell Survival , Crystallography, X-Ray , DNA, Complementary/metabolism , Electrons , Glutathione Transferase/metabolism , Humans , Lymphotoxin beta Receptor , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Fusion Proteins/metabolismABSTRACT
TRAFs (tumor necrosis factor receptor [TNFR]-associated factors) bind to the cytoplasmic portion of liganded TNFRs and stimulate activation of NF-kappaB or JNK pathways. A modulator of TRAF signaling, TANK, serves as either an enhancer or an inhibitor of TRAF-mediated signaling pathways. The crystal structure of a region of TANK bound to TRAF3 has been determined and compared to a similar CD40/TRAF3 complex. TANK and CD40 bind to the same crevice on TRAF3. The recognition motif PxQxT is presented in a boomerang-like structure in TANK that is markedly different from the hairpin loop that forms in CD40 upon binding to TRAF3. Critical TANK contact residues were confirmed by mutagenesis to be required for binding to TRAF3 or TRAF2. Binding affinity, measured by isothermal titration calorimetry and competition assays, demonstrated that TANK competes with CD40 for the TRAF binding site.
