Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.

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Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins. We have determined the high-resolution NMR structure of the complex between the amino-terminal SH3 domain of GRB2 and a ten amino acid peptide derived from the guanine nucleotide releasing factor Sos. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.

Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological lossof-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.cell signaling | phosphorylation | protein kinases | protein-protein interactions | surface receptors L igand-induced tyrosine autophosphorylation plays an important role in the control of activation and cell signaling by receptor tyrosine kinases (1-6). Structural and biochemical studies have shown that autophosphorylation of fibroblast growth factor receptor 1 (FGFR1) (7,8) and FGFR2 (9) are mediated by a sequential and precisely ordered intermolecular reaction that can be divided into three phases. The first phase involves transphosphorylation of a tyrosine located in the activation loop (Y653 in FGFR1) of the catalytic core resulting in 50-100-fold stimulation of kinase activity (7). In the second phase, tyrosine residues that serve as docking sites for signaling proteins are phosphorylated including tyrosines in the kinase insert region (Y583, Y585), the juxtamembrane region (Y463), and in the C-terminal tail (Y766) of FGFR1. In the final and third phase, Y654, a second tyrosine located in the activation loop is phosphorylated, resulting in an additional 10-fold increase in FGFR1 kinase activity (7). Interestingly, tyrosines that are adjacent to one another (e.g. Y653, Y654 and Y583, Y585) are not phosphorylated sequentially, suggesting that both sequence and structural specificities dictate the order of phosphorylation. Although tyrosine phosphorylation plays a major role in cell signaling, it is not yet clear what the structural basis for transautophosphorylation is. In other words, the molecular mechanism underlying how one kinase (the enzyme) within the dimerized receptor specifically and sequentially catalyzes phosphorylation of tyrosine(s) of the other kinase (the substrate) is not yet resolved.We previously determined the crystal structure of activated FGFR1 kinase domain bound to a phospholipase Cγ (PLCγ) fragment composed of two SH2 domains and a tyrosine phosphorylation site (Fig. 1) (PDB code 3GQI) (10). In this s...

ADP ribosylation factors (ARFs), which are members of the Ras superfamily of GTP-binding proteins, are critical components of vesicular trafficking pathways in eukaryotes. Like Ras, ARFs are active in their GTPbound form, and their duration of activity is controlled by GTPase-activating proteins (GAPs), which assist ARFs in hydrolyzing GTP to GDP. PAPβ, a protein that binds to and is phosphorylated by the non-receptor tyrosine kinase PYK2, contains several modular signaling domains including a pleckstrin homology domain, an SH3 domain, ankyrin repeats and an ARF-GAP domain. Sequences of ARF-GAP domains show no recognizable similarity to those of other GAPs, and contain a characteristic Cys-X 2 -Cys-X 16-17 -Cys-X 2 -Cys motif. The crystal structure of the PAPβ ARF-GAP domain and the C-terminal ankyrin repeats has been determined at 2.1 Å resolution. The ARF-GAP domain comprises a central three-stranded β-sheet flanked by five α-helices, with a Zn 2⍣ ion coordinated by the four cysteines of the cysteine-rich motif. Four ankyrin repeats are also present, the first two of which form an extensive interface with the ARF-GAP domain. An invariant arginine and several nearby hydrophobic residues are solvent exposed and are predicted to be the site of interaction with ARFs. Site-directed mutagenesis of these residues confirms their importance in ARF-GAP activity.

Vav is a guanine nucleotide exchange factor for the Rho/Rac family that is expressed exclusively in hematopoietic cells. Growth factor receptor-bound protein 2 (Grb2) has been proposed to play important roles in the membrane localization and activation of Vav through dimerization of its C-terminal Srchomology 3 (SH3) domain (GrbS) and the N-terminal SH3 domain of Vav (VavS). The crystal structure of VavS complexed with GrbS has been solved. VavS is distinct from other SH3 domain proteins in that its binding site for proline-rich peptides is blocked by its own RT loop. One of the ends of the VavS b-barrel forms a concave hydrophobic surface. The GrbS components make a contiguous complementary interface with the VavS surface. The binding site of GrbS for VavS partially overlaps with the canonical binding site for proline-rich peptides, but is de®nitely different. Mutations at the interface caused a decrease in the binding af®nity of VavS for GrbS by 4-to 40-fold. The structure reveals how GrbS discriminates VavS speci®cally from other signaling molecules without binding to the proline-rich motif. Keywords: crystal structure/growth factor receptorbound protein 2/protein±protein interaction/Srchomology 3 domain/Vav

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