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G Gish

Publications and source records attributed to G Gish.

16 recordsLinked to original sources
PubMed1995

A single point mutation switches the specificity of group III Src homology (SH) 2 domains to that of group I SH2 domains.

Src homology 2 (SH2) domains recognize phosphotyrosine-containing sequences, and thereby mediate the association of specific signaling proteins in response to tyrosine phosphorylation (Pawson, T., and Schlessinger, J. (1993) Curr. Biol. 3, 434-442). We have shown that specific binding of SH2 domains to tyrosine-phosphorylated sites is determined by sequences adjacent to the phosphotyrosine. Based on the phosphopeptide specificity and crystal structures, SH2 domains were classified into four different groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778). The beta D5 residues of SH2 domains were predicted to be critical in distinguishing these groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778; Eck, M. J., Shoelson, S. E., and Harrison, S. C. (1993) Nature 362, 87-91). We report here that replacing the aliphatic residues at the beta D5 positions of two Group III SH2 domains (phosphoinositide 3-kinase N-terminal SH2 domain and phospholipase C-gamma C-terminal SH2 domain) with Tyr (as found in Group I SH2 domains) results in a switch in phosphopeptide selectivity, consistent with the specificities of Group I SH2 domains. These results establish the importance of the beta D5 residue in determining specificities of SH2 domains.

Amino Acid Sequence↗
PubMed1995

Construction of an SH2 domain-binding site with mixed specificity.

SH2 domains bind to specific phosphotyrosine-containing sites in a fashion dictated by the amino acids flanking the phosphotyrosine. Attention has focused on the role of the three COOH-terminal positions (+1 to +3) in generating specificity. Autophosphorylation of Tyr1021 in the tail of the beta-receptor for platelet-derived growth factor creates a specific binding site for the COOH-terminal SH2 domain of phospholipase C (PLC)-gamma 1. We show that the residues 4 and 5 amino acids COOH-terminal to Tyr1021 (+4 Leu and +5 Pro) are required for efficient PLC-gamma 1 binding, and that their replacement with the corresponding residues from a phosphatidylinositol 3'-kinase binding site abrogates stable association with PLC-gamma 1. In contrast, replacement of the +3 Pro with Met produces a Tyr1021 site with mixed specificity that binds both PLC-gamma 1 and phosphatidylinositol 3'-kinase. This motif is rendered specific for phosphatidylinositol 3'-kinase by further substitution of the +4 Leu. These results indicate that the +4 and +5 residues are important for the selective binding of specific SH2 domains. This study suggests that phosphotyrosine sites can be tailored to bind one or more SH2 domains with high affinity, depending on the combination of residues in the +1 to +5 positions.

Amino Acid Sequence↗
PubMed1995

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps.

The gene coding for the tyrosine protein kinase domain of v-fps was subcloned into a plasmid vector expressing glutathione-S-transferase (GST). This new vector expresses a fusion protein in Escherichia coli composed of the kinase domain linked with GST at the N-terminus (GST-kin). A portion of the total expressed protein was soluble upon cell lysis and was purified by affinity chromatography using glutathione cross-linked agarose. GST-kin (M(r) 57,000) is a phosphoprotein as judged by 32P autoradiography, consistent with the known autophosphorylation site within the kinase core [Weinmaster et al. (1984) Cell, 37, 559-568]. Cleavage of the fusion protein with thrombin and purification on phosphocellulose resin yielded the pure kinase domain (M(r) 33,000). The activity of the kinase domain is indistinguishable from that of GST-kin using the peptide substrate EEEIYEEIE, indicating that N-terminal fusion has no effect on the kinase domain. GST-kin phosphorylates a second peptide, EAEIYEAIE, with improved catalytic efficiency. Initial velocity data are consistent with a random bireactant mechanism with no substrate synergism observed in the ternary complex. Steady-state kinetic analyses reveal that this peptide is phosphorylated, with a kcat of 3.6 s-1, a Kpeptide of 500 microM and a KATP of 250 microM. The expression, purification and preliminary kinetic analysis of the kinase domain of v-fps provide the first step in the application of structure-function studies for this oncoprotein.

Amino Acid Sequence↗
PubMed1994

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Amino Acid Sequence↗
PubMed1994

Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed with a high affinity binding peptide.

The solution structure of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLC-gamma 1), in complex with a phosphopeptide corresponding to its Tyr-1021 high affinity binding site on the platelet-derived growth factor receptor, has been determined by nuclear magnetic resonance spectroscopy. The topology of the SH2-phosphopeptide complex is similar to previously reported Src and Lck SH2 complexes. However, the binding site for residues C-terminal to the phosphotyrosine (pTyr) is an extended groove that contacts peptide residues at the +1 to +6 positions relative to the pTyr. This striking difference from Src and Lck reflects the fact that the PLC-gamma 1 complex involves binding of a phosphopeptide with predominantly hydrophobic residues C-terminal to the pTyr and therefore serves as a prototype for a second class of SH2-phosphopeptide interactions.

Amino Acid Sequence↗
PubMed1994

Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interactions with cytoplasmic proteins that mediate Ras activation. Such polypeptides include Grb2, which comprises a single Src homology 2 (SH2) domain flanked by two SH3 domains, and the 66, 52 and 46 kDa Shc proteins which possess an SH2 domain in their carboxy-terminus. Grb2 associates with tyrosine phosphorylated proteins through its SH2 domain, and with the Ras guanine nucleotide releasing protein mSos1 through its SH3 domains. mSos1 stimulates conversion of the inactive GDP-bound form of Ras to the active GTP-bound state. In bcr-abl-transformed cells, Grb2 and mSos1 formed a physical complex with Bcr-Abl. In vitro, the Grb2 SH2 domain bound Bcr-Abl through recognition of a tyrosine phosphorylation site within the amino-terminal bcr-encoded sequence (p.Tyr177-Val-Asn-Val), that is common to both Bcr-Abl proteins. These results suggest that autophosphorylation within the Bcr element of Bcr-Abl creates a direct physical link to Grb2-mSos1, and potentially to the Ras pathway, and thereby modifies the target specificity of the Abl tyrosine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗
PubMed1993

SH2 domains recognize specific phosphopeptide sequences.

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

Amino Acid Sequence↗
PubMed1993

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

Amino Acid Sequence↗
PubMed1993

Regulation of c-Src tyrosine kinase activity by the Src SH2 domain.

The protein-tyrosine kinase activity of pp60c-src (c-Src) is inhibited by phosphorylation of tyr527, within the c-Src c-terminal tail. Genetic and biochemical data have suggested that this negative regulation requires an intact Src homology 2 (SH2) domain. Since SH2 domains recognize phosphotyrosine, it is possible that these two non-catalytic domains associate, and thereby repress c-Src kinase activity. Consistent with this model, an isolated Src SH2 domain expressed in bacteria as a GST fusion protein bound in vitro to a synthetic phosphotyrosine-containing peptide modeled on the C-terminal 13 residues of the c-Src tail. Binding was absolutely dependent on phosphorylation of tyr527 in the tail peptide, and was modified by both the length and sequence of the peptide. Competition experiments indicated only a moderate binding affinity between the Src SH2 domain and the phosphorylated tail. A distinct phosphotyrosine-containing peptide previously identified as binding the Src SH2 domain with high affinity stimulated c-Src tyrosine kinase activity in vitro, possibly by competing with the endogenous tail phosphorylation site for binding to the SH2 domain. Indeed, this activation was competitively inhibited by purified bacterial Src SH2 domain. These data provide direct evidence that the c-Src tail has an intrinsic affinity for the Src SH2 domain, and suggest that such an interaction in the intact molecule contributes to maintaining c-Src in an inactive form.

3T3 Cells↗
PubMed1993

Identification of residues in the beta platelet-derived growth factor receptor that confer specificity for binding to phospholipase C-gamma 1.

The SH2 domains of cytoplasmic signaling proteins bind to autophosphorylated growth factor receptors by direct recognition of specific phosphotyrosine-containing sites. To identify the phosphotyrosine involved in association of phospholipase C (PLC)-gamma 1 with the beta platelet-derived growth factor receptor (PDGFR), and to investigate which contiguous residues confer specificity for PLC-gamma 1, phosphotyrosine-containing glutathione S-transferase (GST) fusion proteins possessing different regions of the beta-PDGFR were incubated with lysates of Rat-2 cells that overexpress PLC-gamma 1. The phosphorylated C-terminal tail of the PDGFR bound PLC-gamma 1, but did not associate with phosphatidylinositol (PI) 3'-kinase or GTPase-activating protein (GAP). High-affinity binding of PLC-gamma 1 was dependent on phosphorylation of Tyr-1021. Creation of a new phosphorylation site by replacing Asp-1018 with tyrosine did not restore binding of PLC-gamma 1 in the absence of Tyr-1021, indicating that the location of the phosphorylated tyrosine is important for PLC-gamma 1 binding. Substitution of the proline at the +3 position relative to Tyr-1021 with methionine (Y1021IIP-->Y1021IIM) in the phosphorylated PDGFR tail did not alter PLC-gamma 1 association, but conferred binding activity towards PI 3'-kinase, indicating that this residue is critical in discriminating between PLC-gamma 1 and PI 3'-kinase. Progressive conversion of the three residues C-terminal to Tyr-1021 to the consensus for PI 3'-kinase binding (YMDM) allowed PI 3'-kinase association, but did not block PLC-gamma 1 binding, suggesting that additional residues other than the three residues immediately following the phosphotyrosine may contribute to the association of PLC-gamma 1 with the PDGFR. These results indicate that phosphorylation at Tyr-1021 in the tail of the PDGFR creates a specific binding site for PLC-gamma 1. Proline at the +3 position relative to Tyr-1021 is crucial in conferring specificity for binding to PLC-gamma 1.

Amino Acid Sequence↗
PubMed1989

Phosphorothioates in molecular biology.

The observation that phosphorothioate analogues of the nucleoside triphosphates are substrates for DNA- and RNA-polymerases has proven a boon for the molecular biologist. As these phosphorothioate-containing polymers are stable to degradation by nucleases and the sulfur atom confers many favourable chemical properties, several applications in molecular biology have been developed, including new methods for site-directed mutagenesis and DNA sequencing.

DNA↗
PubMed1988

Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates.

The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described. The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-O-(1-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be used to obtain the same amount of degradation as with 2-iodoethanol.

Base Sequence↗
PubMed1988

DNA and RNA sequence determination based on phosphorothioate chemistry.

The difference in reactivity between phosphate and phosphorothioate diesters is the basis of a chemical degradation scheme for the sequencing of DNA and RNA. The phosphorothioate groups are incorporated into the nucleic acid in four separate enzymatic reactions, with three of the natural nucleoside triphosphates and one alpha-thiotriphosphate in each reaction. Selective strand cleavage is achieved through alkylation to form the hydrolytically labile phosphorothioate triester. As an example, the sequence analysis is presented of M13 phage DNA and of RNA prepared by transcription with SP6 RNA polymerase.

Base Sequence↗
PubMed1987

DNA and RNA sequencing utilizing phosphorothioate chemistry.

A method for nucleic acid sequencing has been developed based on the observation that phosphorothioate diesters are hydrolysed by treatment with 2-iodoethanol in a solution of aqueous ethanol. For DNA sequencing, primed single-stranded M13 DNA is polymerised with the Klenow fragment of DNA polymerase I in the presence of the three normal deoxyribonucleotide triphosphates and one alpha-phosphorothioate derivative. This is followed by treatment with 2-iodoethanol, precipitation of the DNA fragments and analysis by polyacrylamide electrophoresis. RNA transcribed from plasmids containing the SP6 RNA polymerase promoter is sequenced by including the alpha-phosphorothioate derivative of the ribonucleotide triphosphates in the polymerisation and treating the product with iodoethane. The cleavage reaction involves alkylation of the sulfur atom to form the phosphorothioate triester and hydrolysis catalysed by an adjacent hydroxyl group.

Base Sequence↗
PubMed1984

Interaction of thiamin diphosphate and thiamin thiazolone diphosphate with wheat germ pyruvate decarboxylase.

The interactions of the apoenzyme of wheat germ pyruvate decarboxylase with thiamin diphosphate and with thiamin thiazolone diphosphate have been investigated. The results test hypotheses concerning the structure of the transition state for decarboxylation of the enzyme-bound adduct of pyruvate and thiamin diphosphate. Thiamin thiazolone diphosphate, a possible transition state analogue, binds to the apoenzyme by a two-step process. The first is slow and reversible (k = 200 M-1 s-1; K = 5 X 10(-7) M). The second step is irreversible (k = 1 X 10(-6) s-1). The rate constant for activation by thiamin diphosphate is 160 M-1 s-1. Thiamin diphosphate is released very slowly from the holoenzyme (k = 2 X 10(-5) s-1). Thiamin thiazolone diphosphate competitively inhibits activation of the apoenzyme by thiamin diphosphate, Ki = 2 X 10(-6) M. Km for thiamin diphosphate is only 3 times larger. Thiamin thiazolone is solvated preferentially to thiamin in 2-butanol, a medium whose polarity should resemble that of the binding site. It is concluded that the observed high affinity of thiamin thiazolone diphosphate for the apoenzyme is the result of a combination of effects which do not require the assumption that it is an analogue of the transition state for the decarboxylation of enzyme-bound 2-(2-lactyl) thiamin diphosphate.

Carboxy-Lyases↗