Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF.

Nerve growth factor (NGF) is a neurotrophic factor responsible for the differentiation and survival of sympathetic and sensory neurons as well as selective populations of cholinergic neurons. NGF binds to specific cell-surface receptors but the mechanism for transduction of the neurotrophic signal is unknown. Several experiments using the NGF-responsive pheochromocytoma cell line, PC12, have implicated tyrosine phosphorylation in NGF-mediated responses, although no NGF-specific tyrosine kinases have been identified. Here we show that NGF induces tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product, a tyrosine kinase receptor whose expression is restricted in vivo to neurons of the sensory spinal and cranial ganglia of neural crest origin. Tyrosine phosphorylation of trk by NGF is rapid, specific and occurs with picomolar quantities of factor, indicating that the response is mediated by physiological amounts of NGF. Activation of the trk tyrosine kinase receptor provides a possible mechanism for signal transduction by NGF.     

Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.

Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.     

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.     

SH2域与磷酸化多肽相互作用的连续溶剂模型研究

使用连续溶剂模型方法研究了SH2结构域与磷酸化多肽pYXXX(X为20种常见氨基酸残基中的任意一种)之间的相互作用.首先计算了已知的SH2域-磷酸化酪氨酸多肽复合物之间的结合能,理论计算的结果与实验测得的亲合能之间的相关系数为0.91,验证了理论模型的正确性.然后,用该模型方法计算了SH2域与pYXXX之间的结合能,分析了磷酸化酪氨酸多肽pYXXX中 1, 2, 3位置上残基对结合能的影响.结果表明 2, 3位置上残基的变化对结合能影响较大, 2位置上带负电的残基和 3位置上的疏水性残基有利于SH2域与pYXXX之间的相互作用,这与实验结果一致.     

Crystal structure of a Src-homology 3 (SH3) domain.

The Src-homologous SH3 domain is a small domain present in a large number of proteins that are involved in signal transduction, such as the Src protein tyrosine kinase, or in membrane-cytoskeleton interactions, but the function of SH3 is still unknown (reviewed in refs 1-3). Here we report the three-dimensional structure at 1.8 A resolution of the SH3 domain of the cytoskeletal protein spectrin expressed in Escherichia coli. The domain is a compact beta-barrel made of five antiparallel beta-strands. The amino acids that are conserved in the SH3 sequences are located close to each other on one side of the molecule. This surface is rich in aromatic and carboxylic amino acids, and is distal to the region of the molecule where the N and C termini reside and where SH3 inserts into the alpha-spectrin chain. We suggest that a protein ligand binds to this conserved surface of SH3.     

Cloning and domain structure of the mammalian splicing factor U2AF.

A complementary DNA clone encoding the large subunit of the essential mammalian pre-messenger RNA splicing component U2 snRNP auxiliary factor (U2AF65) has been isolated and expressed in vitro. It contains two functional domains: a sequence-specific RNA-binding region composed of three ribonucleoprotein-consensus sequence domains, and an arginine/serine-rich motif necessary for splicing but not for binding to pre-mRNA.     

Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase.

Receptor protein-tyrosine kinases, through phosphorylation of specific tyrosine residues, generate high-affinity binding sites which direct assembly of multienzyme signalling complexes. Many of these signalling proteins, including phospholipase C gamma, GTPase-activating protein and phosphatidylinositol-3-OH kinase, contain src-homology 2 (SH2) domains, which bind with high affinity and specificity to tyrosine-phosphorylated sequences. The critical role played by SH2 domains in signalling has been highlighted by recent studies showing that mutation of specific phosphorylation sites on the platelet-derived growth factor receptor impair its association with phosphatidylinositol-3-OH kinase, preventing growth factor-induced mitogenesis. Here we report the solution structure of an isolated SH2 domain from the 85K regulatory subunit of phosphatidylinositol-3-OH kinase, determined using multidimensional nuclear magnetic resonance spectroscopy. The structure is characterized by a central region of beta-sheet flanked by two alpha-helices, with a highly flexible loop close to functionally important residues previously identified by site-directed mutagenesis.     

Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis

The cdc2+ protein kinase (pp34) is found to be phosphorylated on tyrosine as well as serine and threonine residues in exponentially growing Schizosaccharomyces pombe. At mitosis, the level of pp34 phosphorylation on both threonine and tyrosine residues decreases. The single detectable site of tyrosine phosphorylation in pp34 has been mapped to Tyr 15, a residue within the presumptive ATP-binding domain. Substitution of this tyrosine by phenylalanine advances cells prematurely into mitosis, establishing that tyrosine phosphorylation/dephosphorylation directly regulates pp34 function.     

C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains.

The induction of the hermaphrodite vulva and the migration of the sex myoblasts in the nematode Caenorhabditis elegans are both controlled by intercellular signalling. The gonadal anchor cell induces formation of the vulva from nearby hypodermal cells, and a set of somatic gonadal cells attract the migrating sex myoblasts to their final positions. Many genes required for vulval induction have been identified, including the let-23 receptor tyrosine kinase gene and the let-60 ras gene. We report here the identification and characterization of a new gene, sem-5 (sem, sex muscle abnormal), that acts both in vulval induction and in sex myoblast migration. On the basis of its DNA sequence, sem-5 encodes a novel 228-amino-acid protein which consists almost entirely of one SH2 (SH, src homology region) and two SH3 domains. SH2 and SH3 domains are present in many signalling proteins regulated by receptor and non-receptor tyrosine kinases. Mutations that impair sem-5 activity alter residues that are highly conserved among different SH2 and SH3 domains. Our results indicate that the sem-5 gene encodes a novel protein that functions in at least two distinct cell-signalling processes.