PubMed Health⌕ Search

Biomedical subjects

J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 91 records · Page 5Linked to original sources
PubMed1996

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Animals↗
PubMed1996

The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1.

Nck is an adaptor protein composed of a single SH2 domain and three SH3 domains. Upon growth factor stimulation, Nck is recruited to receptor tyrosine kinases via its SH2 domain, probably initiating one or more signaling cascades. In this report, we show that Nck is bound in living cells to the serine-threonine kinase Pak1. The association between Nck and Pak1 is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of Pak1. We also show that Pak1 is recruited by activated epidermal growth factor (EGF) and platelet-derived growth factor receptors. Moreover, Pak1 kinase activity is increased in response to EGF in HeLa cells transfected with human Pak1, and the kinase activity was enhanced when Nck was co-transfected. It is concluded that Nck links receptor tyrosine kinases with Pak1 and is probably involved in targeting and regulation of Pak1 activity.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism.

The crystal structure of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1K) has been determined in its unliganded form to 2.0 angstroms resolution and in complex with with an ATP analog to 2.3 angstrosms A resolution. Several features distinguish the structure of FGFR1K from that of the tyrosine kinase domain of the insulin receptor. Residues in the activation loop of FGFR1K appear to interfere with substrate peptide binding but not with ATP binding, revealing a second and perhaps more general autoinhibitory mechanism for receptor tyrosine kinases. In addition, a dimeric form of FGFR1K observed in the crystal structure may provide insights into the molecular mechanisms by which FGF receptors are activated. Finally, the structure provides a basis for rationalizing the effects of kinase mutations in FGF receptors that lead to developmental disorders in nematodes and humans.

Adenosine Triphosphate↗
PubMed1996

Activation of Pyk2 by stress signals and coupling with JNK signaling pathway.

The c-Jun amino-terminal kinase (JNK) is activated by various heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors, inflammatory cytokines, and stress signals. Yet, upstream mediators that link extracellular signals with the JNK signaling pathway are currently unknown. The tyrosine kinase Pyk2 was activated by tumor necrosis factor alpha, by ultraviolet irradiation, and by changes in osmolarity. Overexpression of Pyk2 led to activation of JNK, and a dominant-negative mutant of Pyk2 interfered with ultraviolet light- or osmotic shock-induced activation of JNK. Pyk2 represents a cell type-specific, stress-sensitive mediator of the JNK signaling pathway.

Animals↗
PubMed1996

Three forms of RPTP-beta are differentially expressed during gliogenesis in the developing rat brain and during glial cell differentiation in culture.

In situ hybridization and Northern analysis demonstrate that the three splicing variants of RPTP-beta have different spatial and temporal patterns of expression in the developing brain. The 9.5-kb and 6.4-kb transcripts, which encode transmembrane protein tyrosine phosphatases with different extracellular domains, are predominantly expressed in glial progenitors located in the subventricular zone (SVZ). The 8.4-kb transcript, which encodes a secreted chondroitin sulfate proteoglycan (phosphacan), is expressed at high levels by more mature glia that have migrated out of the SVZ. The three transcripts are also differentially expressed in glial cell cultures; O2A progenitors express high levels of the 9.5- and 8.4-kb transcript, whereas type 1 astrocyte progenitors predominantly express the 6.4-kb transcript. C6 gliomas also express high levels of the 6.4-kb transcript. Treating C6 cells with the differentiating agent dibutyryl cyclic-AMP (DBcAMP), induces a decrease in the 6.4-kb transcript and a corresponding increase in the 8.4-kb transcript. O2A cells grown in the presence of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) remain highly proliferative and undifferentiated, and continue to express high levels of RPTP-beta. However, when O2A cells are grown in conditions that induce oligodendrocyte differentiation, there is a marked decrease in the expression of the transmembrane forms of RPTP-beta, as determined by immunofluorescence. These results demonstrate that RPTP-beta expression is regulated during glial cell differentiation and suggest that the different forms of RPTP-beta perform distinct functions during brain development.

Animals↗
PubMed1996

Thermodynamic studies of SHC phosphotyrosine interaction domain recognition of the NPXpY motif.

The N-terminal 200 amino acids of SHC constitute a unique phosphotyrosine (Tyr(P)) interaction (PI) domain that shows no significant sequence similarity to the other Tyr(P)-recognizing module, the SH2 domain. We describe the thermodynamic parameters characterizing PI domain binding to various tyrosyl phosphopeptides, using isothermal titration calorimetry. The PI domain forms 1:1 complexes of similar affinity with a 12-mer peptide (ISLDNPDpYQQDF) derived from Tyr-1148 of the epidermal growth factor receptor (EGFR) (KD = 28 nm) and an 18-mer (LQGHIIENPQpYFSDACVH) derived from Tyr-490 of Trk (KD = 42 nM). Binding of the EGFR-derived peptide was largely enthalpy-driven at 25 degrees C, while Trk490 peptide binding was entropy-driven. Based on the change in heat capacity upon binding, approximately 700 A2 of nonpolar surface was estimated to be buried upon interaction. Alteration of the Asn or Pro to Ala in the NPXpY motif of the EGFR Tyr-1148 peptide increased the KD of PI domain interactions to 238 and 370 nM, respectively. Alteration of a Leu at position -5 (with respect to Tyr(P)) in the EGFR peptide to Gly also reduced the binding affinity (KD = 580 nM). It is proposed that the PI domain recognizes the beta1 turn that is found in NPXpY-containing peptides and also interacts with a larger segment of the peptide than seen for SH2 domains.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.

It has recently been suggested that pleckstrin homology (PH) domains bind specifically to phospholipids, with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) being most strongly bound. This observation suggests that PH domains may be responsible for membrane association of proteins in which they occur. Further, this membrane association may be regulated by enzymes that modify lipid head groups to which PH domains may bind. We have studied the binding of phospholipids to the PH domain of human dynamin, a 100 kDa GTPase that is involved in the initial stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We confirm that PtdIns(4,5)P2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a specific site on the surface of dynamin PH domain of which binding of gIns(1,4,5)P3 (the head-group skeleton of PtdIns(4,5)P2) occurs. The relative affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, therefore, that the interaction of these phospholipids with dynamin is likely to occur via the PH domain. Given the fact that PH domains are often found in proteins associated with GTPase activity, or in guanine nucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis.

Binding Sites↗
PubMed1996

Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes.

Crystal structures of the amino-terminal SH2 domain of the p85alpha subunit of phosphatidylinositol (PI) 3-kinase, alone and in complex with phosphopeptides bearing pTyr-Met/Val-Xaa-Met motifs, show that phosphopeptides bind in the two-pronged manner seen in high-affinity Lck and Src SH2 complexes, with conserved interactions between the domain and the peptide segment from phosphotyrosine to Met+3. Peptide binding requires the rearrangement of a tyrosyl side chain in the BG loop to create the hydrophobic Met+3 binding pocket. The structures suggest a mechanism for the biological specificity exhibited by PI 3-kinase in its interactions with phosphoprotein partners.

Amino Acid Sequence↗
PubMed1996

Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor.

The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells.

Base Sequence↗
PubMed1996

A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

Amino Acid Sequence↗
PubMed1996

Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction.

Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.

Amino Acid Sequence↗
PubMed1996

The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.

Amino Acid Sequence↗
PubMed1995

Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain.

The X-ray crystal structure of the high affinity complex between the pleckstrin homology (PH) domain from rat phospholipase C-delta 1 (PLC-delta 1) and inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P3) has been refined to 1.9 A resolution. The domain fold is similar to others of known structure. Ins(1,4,5)P3 binds on the positively charged face of the electrostatically polarized domain, interacting predominantly with the beta 1/beta 2 and beta 3/beta 4 loops. The 4- and 5-phosphate groups of Ins(1,4,5)P3 interact much more extensively than the 1-phosphate. Two amino acids in the PLC-delta 1 PH domain that contact Ins(1,4,5)P3 have counterparts in the Bruton's tyrosine kinase (Btk) PH domain, where mutational changes cause inherited agammaglobulinemia, suggesting a mechanism for loss of function in Btk mutants. Using electrostatics and varying levels of head-group specificity, PH domains may localize and orient signaling proteins, providing a general membrane targeting and regulatory function.

Amino Acid Sequence↗
PubMed1995

Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been reported. A region that includes the PH domain has also been implicated in binding of phospholipase C-delta 1 (PLC-delta 1) to both PtdIns(4,5)P2 and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [Cifuentes, M. E., Delaney, T. & Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948]. We report herein that the isolated PH domain from PLC-delta 1 binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 with high affinity and shows the same binding specificity seen by others with whole PLC-delta 1. Thus the PH domain is functionally and structurally modular. These results demonstrate stereo-specific high-affinity binding by an isolated PH domain and further support a functional role for PH domains in the regulation of PLC isoforms. Other PH domains did not bind strongly to the compounds tested, suggesting that inositol phosphates and phospholipids are not likely physiological ligands for all PH domains. Nonetheless, since all PH-domain-containing proteins are associated with membrane surfaces, several PH domains bind to specific sites on membranes, and PH domains appear to be electrostatically polarized, a possible general role for PH domains in membrane association is suggested.

Amino Acid Sequence↗
PubMed1995

Signal transduction by the alpha 6 beta 4 integrin: distinct beta 4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes.

We have examined the mechanism of signal transduction by the hemidesmosomal integrin alpha 6 beta 4, a laminin receptor involved in morphogenesis and tumor progression. Immunoprecipitation and immune complex kinase assays indicated that antibody- or laminin-induced ligation of alpha 6 beta 4 causes tyrosine phosphorylation of the beta 4 subunit in intact cells and that this event is mediated by a protein kinase(s) physically associated with the integrin. Co-immunoprecipitation and GST fusion protein binding experiments showed that the adaptor protein Shc forms a complex with the tyrosine-phosphorylated beta 4 subunit. Shc is then phosphorylated on tyrosine residues and recruits the adaptor Grb2, thereby potentially linking alpha 6 beta 4 to the ras pathway. The beta 4 subunit was found to be phosphorylated at multiple tyrosine residues in vivo, including a tyrosine-based activation motif (TAM) resembling those found in T and B cell receptors. Phenylalanine substitutions at the beta 4 TAM disrupted association of alpha 6 beta 4 with hemidesmosomes, but did not interfere with tyrosine phosphorylation of Shc and recruitment of Grb2. These results indicate that signal transduction by the alpha 6 beta 4 integrin is mediated by an associated tyrosine kinase and that phosphorylation of distinct sites in the beta 4 tail mediates assembly of the hemidesmosomal cytoskeleton and recruitment of Shc/Grb2.

Adaptor Proteins, Signal Transducing↗