Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases.

The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction.     

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.     

Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I

In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. We have now cloned and sequenced the gene encoding ABP1p. ABP1p is a novel protein with a 50 amino-acid C-terminal domain that is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. We also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton.     

Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases.

The mammalian shc gene encodes two overlapping, widely expressed proteins of 46 and 52K, with a carboxy-terminal SH2 domain that binds activated growth factor receptors, and a more amino-terminal glycine/proline-rich region. These shc gene products (Shc) are transforming when overexpressed in fibroblasts. Shc proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors, and in cells transformed by v-src (ref. 2), suggesting that they are tyrosine kinase targets that control a mitogenic signalling pathway. Here we report that tyrosine-phosphorylated Shc proteins form a specific complex with a non-phosphorylated 23K polypeptide encoded by the grb2/sem-5 gene. The grb2/sem-5 gene product itself contains an SH2 domain, which mediates binding to Shc, and is implicated in activation of the Ras guanine nucleotide-binding protein by tyrosine kinases in both Caenorhabditis elegans and mammalian cells. Consistent with a role in signalling through Ras, shc overexpression induced Ras-dependent neurite outgrowth in PC12 cells. These results suggest that Shc tyrosine phosphorylation can couple tyrosine kinases to Grb2/Sem-5, through formation of a Shc-Grb2/Sem-5 complex, and thereby regulate the mammalian Ras signalling pathway.     

Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation

Tyrosine-specific protein phosphorylation has been implicated in the regulation of cell transformation and proliferation. However, recent studies have shown that the expression of protein tyrosine kinases in adult brain is very high, suggesting that tyrosine-specific protein phosphorylation may also have a role in the regulation of neuronal function. Although a number of substrate proteins are phosphorylated on tyrosine residues, the functional alteration of proteins by tyrosine phosphorylation has previously been convincingly demonstrated only for protein tyrosine kinases. The nicotinic acetylcholine receptor, a neurotransmitter-gated ion channel, is phosphorylated by a protein tyrosine kinase in post-synaptic membranes in vitro and in vivo. We demonstrate here that this tyrosine phosphorylation increases the rate of the rapid phase of desensitization of the nicotinic receptor, as measured by single channel recording of purified nicotinic acetylcholine receptor, when reconstituted in lipid vesicles. These data provide direct evidence for the regulation of ion channel properties by tyrosine phosphorylation. The results, which demonstrate a functional role of tyrosine phosphorylation in the nervous system, suggest a widespread role for tyrosine phosphorylation in neuronal signal transduction.     

The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes.     

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.     

3BP2的SH2结构域的晶体结构和与来源于FRS2的肽的复合物的晶体结构

3BP2最初被作为一个Abl SH3结合蛋白被分离,但是其功能并不确定。除了富含脯氨酸区域和间接与SH3结合外,3BP2还有一个PH和Src同源区2结构域(SH2)。 Src同源区2结构域(SH2)是一个很大的家族,它们通过人体基因组编码的模块间的相互作用来识别酪氨酸磷酸化序列,由此在细胞信号转导和控制中发挥中心作用。肽基可以被SH2识别从而形成一种复合物.这篇文章的内容是关于3BP2的SH2结构域的晶体结构和与来源于FRS2的肽的复合物的晶体结构。依照表面电荷性质,这个结合袋的特异性是半极性半中性的。这个结构的特点明显的表现在,对于亲和性来说,Glu(p+1)比Ala (p+1) 或 Val (p+1)更为重要。     

LysM蛋白在植物免疫中的作用

溶解素基序（LysM）是在多种蛋白质中普遍存在的结构域.植物LysM蛋白能够感知几丁质及其寡糖等分子配体，从而启动植物对病原菌的免疫反应.在水稻、拟南芥等植物免疫应答过程中，LysM蛋白作为一种重要的模式识别受体，通过不同形式的寡聚化，激活多种类受体胞质激酶及其下游的MAPK（mitogen activated protein kinase）级联反应传递信号.同时，蛋白质可逆磷酸化和蛋白质降解途径可以负调节LysM蛋白介导的防御信号转导.文章综述了植物免疫过程中LysM蛋白介导的信号转导分子机制.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis.

Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis.     

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.     

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.     

Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.     

Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases.

Mitogen activated protein (MAP) kinases (MAPKs) are a family of protein-serine/threonine kinases activated as an early intracellular response to a variety of hormones and growth factors. They are unique in requiring both serine/threonine and tyrosine phosphorylation to become active and are the only examples of protein-serine/threonine kinases activated by tyrosine phosphorylation. Nerve growth factor (NGF) promotes differentiation of phaeochromocytoma (PC12) cells, which respond by conversion within hours from a chromaffin-like to a sympathetic neuron-like phenotype. NGF stimulation of PC12 cells increases the activity of two protein kinases by greater than 20-fold within minutes, both strikingly similar to MAPKs. They are inactivated by either protein-tyrosine phosphatases or the protein-serine/threonine phosphatase termed protein phosphatase 2A (ref. 8), they activate protein S6 kinase-II (refs 9, 10), and they phosphorylate identical threonine residues on myelin basic protein (our unpublished results) to those phosphorylated by other MAPKs. Immunological data indicate that these protein kinases, termed peak-I and peak-II (Fig. 1a) are probably ERK2 and ERK1, respectively, two widely expressed MAPK isoforms. Here we identify the 'MAP kinase kinases' (MAPKKs) in PC12 cells which are activated by NGF and report that MAPKKs are dependent on serine/threonine phosphorylation for activity and promote phosphorylation of serine/threonine and tyrosine residues on MAPKs.     

PDGF induction of tyrosine phosphorylation of GTPase activating protein

The cascade of biochemical events triggered by growth factors and their receptors is central to understanding normal cell-growth regulation and its subversion in cancer. Ras proteins (p21ras) have been implicated in signal transduction pathways used by several growth factors, including platelet-derived growth factor (PDGF). These guanine nucleotide-binding Ras proteins specifically interact with a cellular GTPase-activating protein (GAP). Here we report that in intact quiescent fibroblasts, both AA and BB homodimers of PDGF rapidly induce tyrosine phosphorylation of GAP under conditions in which insulin and basic fibroblast growth factor (bFGF) are ineffective. Although GAP is located predominantly in the cytosol, most tyrosine-phosphorylated GAP is associated with the cell membrane, the site of p21ras biological activity. These results provide a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signalling system.     

EGFR信号转导机制及靶向治疗

综述了EGFR基本结构特征及其介导细胞信号转导的机制,论述了基于EGFR靶向治疗的机理及研究现状。指出,EGFR是最早被发现并研究的RTK家族成员,其蛋白结构分成胞外域、跨膜区与胞内域三部分。EGFR介导细胞信号转导的核心机制是配体EGF与EGFR胞外域结合,通过变构作用与二聚化作用,使胞内域通过反式激活完成对受体末端酪氨酸残基的磷酸化,进而招募下游信号因子完成细胞信号转导过程。质膜结构与组成、EGFR跨膜区的组成及胞外域的有无、EGFR的内吞及泛素依赖性降解过程都调控EGFR细胞信号转导过程。阻断EGFR信号通路可抑制表皮肿瘤细胞成长。EGFR已经成为表皮肿瘤治疗的重要靶点。     

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.