The Src homology 3 (SH3) region is a protein domain of 55 to 75 amino acids found in many cytoplasmic proteins, including those that participate in signal transduction pathways. The solution structure of the SH3 domain of the tyrosine kinase Src was determined by multidimensional nuclear magnetic resonance methods. The molecule is composed of two short three-stranded anti-parallel beta sheets packed together at approximately right angles. Studies of the SH3 domain bound to proline-rich peptide ligands revealed a hydrophobic binding site on the surface of the protein that is lined with the side chains of conserved aromatic amino acids.

In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p2l"GAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src-and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with Mrs of 62,000 and 130,000 was inhibited by incubation with a Src S13 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.The Src family of protein tyrosine kinases contains two conserved domains, the Src homology 2 (SH2) and Src homology 3 (SH3) domains. SH2 and SH3 domains mediate intramolecular and intermolecular binding interactions which regulate the functional activity of these proteins in intracellular signal transduction pathways (20,37,38). The intermolecular protein-protein interactions couple Src family kinases with proteins which serve as protein substrates or which determine the subcellular localization of these enzymes (5, 20). The intramolecular binding interactions mediated by SH2 and SH3 domains regulate the catalytic activity of these kinases (20, 50, 56). SH2 and SH3 domains are also found in other protein tyrosine kinases (e.g., Abl, Fps, Syk, and Zap) as well as in cellular proteins otherwise unrelated to Src family kinases (38). These proteins include enzymes (e.g., phospholipase C--y, p21aSGAP [a Ras GTPase-activating protein], and the p85 subunit of phosphatidylinositol 3'-kinase [p85-PI-3K]), transcription factors (e.g., the p113, p91, and p84 subunits of interferon-stimulated gene factor 3), cytoskeletal proteins (e.g., tensin, cortactin, myosin 1B, and a-spectrin), and adaptor proteins that appear to serve exclusively as coupling factors that link proteins involved in signaling pathways (e.g., Grb-2/ Sem5, Nck, and Crk) (6,8,14,20,28,31,37,38 and SH3 domains are commonly found together in intracellular p...

The amino-terminal, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases.The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14-to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p1209, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. pllO was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.

The semaphorin family contains a large number of secreted and transmembrane proteins, some of which are known to act as repulsive axon guidance cues during development or to be involved in immune function. We report here on the identification of semaphorin K1 (sema K1), the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord.The semaphorins constitute a large family of evolutionally conserved glycoproteins that are defined by a characteristic semaphorin domain of approximately 500 amino acids (1-3). The first vertebrate semaphorin, collapsin-1 in chick, was identified by its ability to induce growth cone collapse (4). Consistent with this function, its mammalian homologue, sema III, has been shown to repel specific subsets of sensory axons (5). As a result of these and other studies, Coll-1/sema III/D has been implicated in the patterning of sensory axon projections into the ventral spinal cord and cranial nerve projections into the periphery (6 -11).Several other semaphorins have also been implicated as repulsive and/or attractive cues in axon guidance, axon fasciculation, and synapse formation (1,(12)(13)(14)(15)(16)(17). In addition, members of the semaphorin family have been implicated in functions outside the nervous system, including bone skeleton and heart formation (9), immune function (18, 19), tumor suppression (20 -22), and conferring drug resistance to cells (23).Recent studies have identified the first semaphorin receptor as a member of the neuropilin family. Neuropilin-1 is a high affinity receptor for sema III, E, and IV, whereas neuropilin-2 binds differentially to the subfamily of secreted semaphorins (24 -27).The vertebrate semaphorin family can be classified into several phylogenetically distinct subfamilies (Ref. 15, Fig. 1B). Each subfamily has a unique structural arrangement of protein domains. The secreted members of the semaphorin family contain a characteristic semaphorin domain at the NH 2 terminus, followed by an immunoglobulin (Ig) domain and a stretch of basic amino acids in the carboxyl-terminal region. Between the NH 2 -terminal semaphorin domain and the transmembrane spanning region, the transmembrane semaphorins contain several alternative structural motifs, including either an Ig domain, a stretch of thrombospondin repeats, or a sequence with no obvious domain homology. Interestingly, semaphorin-like sequences have been identifi...

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 M. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.