Biomedical subjects

M D Waterfield

Publications and source records attributed to M D Waterfield.

At least 73 records · Page 4Linked to original sources

PubMed1995

Modulation of the substrate specificity of the mammalian phosphatidylinositol 3-kinase by cholesterol sulfate and sulfatide.

The substrate specificity of the purified, mammalian phosphatidylinositol 3-kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed.

Adenosine Triphosphate↗

PubMed1995

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Amino Acid Sequence↗

PubMed1995

The crystal structure of the N-terminal SH3 domain of Grb2.

The 3-D structure of the N-terminal SH3 domain of the regulatory protein Grb2 has been determined by X-ray analysis at 2.8 A resolution and refined to a crystallographic R factor of 21.5%. The structure, which is very similar to those of other SH3 domains, consists of two orthogonal, antiparallel up-down beta-sheets, with three variable loops and a 3(10) helix. Docking of the proline-rich peptide, 3BP1 on Grb2-N SH3, shows that the polyproline type II helix can bind the SH3 domain forming conserved hydrogen bonds between the main-chain carbonyl oxygens of Met4 and Pro7 of the proline-rich peptide and the reoriented side-chains of Trp36 and Asn51, respectively, and a hydrogen bond between the main-chain carbonyl of Leu8 of the proline rich peptide with the side-chain OH of Tyr52 of the Grb2-N SH3. The peptide side-chain binding occurs on the surface of SH3 domain at three major sites involving the side-chains of the residues in the hydrophobic patch (Tyr7, Phe9, Trp36, Phe47, Pro49 and Tyr52) and the RT-Src and n-Src loops of the SH3 domain. The proline-rich peptides could bind the Grb2-N SH3 in either orientation and maintain the key hydrogen bonds because of the pseudo-symmetry of the polyproline type II helix. However, for the mSos1 peptide a salt bridge can be formed between the arginine of the proline-rich peptide and the protein at Asp15, Glu16 and Glu31 only in one direction; this orientation seems to be strongly preferred. The conservatively varied RGD sequence motif (sometimes KGE or KGD) in SH3 domains might be involved in interactions at the cell membrane.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Ras-independent and wortmannin-sensitive activation of glycogen synthase by insulin in Chinese hamster ovary cells.

Activation of glycogen synthase is one of the major metabolic events triggered by exposure of cells to insulin. The molecular mechanism by which insulin activates glycogen synthase was investigated. The possible role of Ras and mitogen-activated protein kinase cascade was investigated with a stable cell line, CHO-IR-C/S 46, that overexpresses insulin receptors and a catalytically inactive SH-PTP 2 protein phosphatase and in which insulin does not induce the formation of the Ras-GTP complex or the subsequently activation of the mitogen-activated protein kinase cascade. Insulin activated glycogen synthase in this cell line to a similar extent as in parental CHO-IR cells. The importance of heteromeric phosphoinositide (PI) 3-kinase in insulin activation of glycogen synthase was examined in a stable cell line, CHO-IR/delta p85, that overexpresses insulin receptors and a dominant negative mutant (delta p85) of the 85-kDa subunit of PI 3-kinase that lacks the binding site for the catalytic 110-kDa subunit. Insulin-dependent activation of PI-3 kinase and glucose transport, but not the formation of the Ras-GTP complex, are markedly attenuated in this cell line. In CHO-IR/delta p85 cells, insulin activated glycogen synthase to a similar extent as in parental CHO-IR cells. The failure of overproduction of the mutant (delta p85) protein to inhibit insulin activation of glycogen synthase was also confirmed by transient expression in Rat 1 cells with the use of a recombinant vaccinia virus. However, wortmannin abolished insulin activation of glycogen synthase in all cell lines. These data suggest that existence of a Ras-independent and wortmannin-sensitive pathway for activation of glycogen synthase by insulin.

Androstadienes↗

PubMed1995

The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein.

The receptor of Hepatocyte Growth Factor-Scatter Factor (HGF) is a tyrosine kinase which regulates cell motility and growth. After ligand-induced tyrosine phosphorylation, the HGF receptor associates with the Shc adaptor, via the SH2 domain. Site-directed mutagenesis of the HGF receptor indicates that phosphotyrosines Y1349VHV and Y1356VNV can work as docking sites for Shc. The Kd of this interaction, measured in real time using synthetic phosphopeptides and recombinant Shc on a BIAcore biosensor, is 150 nm for both sites. After stimulation of the HGF receptor, Shc is phosphorylated on Y317VNV, generating an high affinity binding site for Grb2 (Kd = 15 nM). This duplicates the high affinity binding site for Grb2 present on the HGF receptor (Y1356VNV). Thus HGF stimulation can trigger the Ras pathway by recruiting Grb2 both directly through the receptor, and indirectly, through Shc. Overexpression of wild-type Shc, but not of the Y317-->F mutant, enhances cell migration and growth in response to HGF. These data show that Shc is a relevant substrate of the HGF receptor, and works as an 'amplifier' of the motogenic as well as of the mitogenic response.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase.

The role of heteromeric phosphoinositide (PI) 3-kinase activity in insulin signal transduction was studied by investigating the effects of (i) overexpression of a dominant negative mutant p85 (delta p85) that lacks the binding site for p110 (delta p85-overexpressing cells) and (ii) inhibition of PI 3-kinase activity by wortmannin (wortmannin-treated cells). The insulin-induced association of PI 3-kinase activity with insulin receptor sustrate-1 (IRS-1) was inhibited in both wortmannin-treated cells and delta p85-overexpressing cells. However, whereas insulin-induced activation of p70 S6 kinase was completely abolished in wortmannin-treated cells, it appeared normal in delta p85-overexpressing cells. These results raise the possibility that a wortmannin-sensitive pathway independent of heteromeric PI 3-kinase is involved in the activation of p70 S6 kinase by insulin.

Amino Acid Sequence↗

PubMed1995

Structural basis for pleckstrin homology domain mutations in X-linked agammaglobulinemia.

Deficiencies in a tyrosine kinase, designated Btk, cause X-linked agammaglobulinemia (XLA) in man, a hereditary defect of B-cell differentiation. Mutations in the newly found PH domain located at the N-terminus of Btk have been shown to be the direct cause of XLA, and here two new mutations, T33P and V64F, are presented. Btk is thus far the only protein in which mutations of the PH domain have been found to cause a disease. The three-dimensional structure of the Btk PH domain was modeled on the basis of the dynamin PH structure. Despite a relatively low sequence similarity the Btk PH domain seems to have the same two beta-sheet structure observed in the known structures. The model was used to interpret the structural basis for disease in five independent point mutations and in an insertion in patients with XLA. The mutated residues F25, V64, and V113, and possibly residue(s) around Q103, could form a binding site, since these amino acids are located close to each other on the surface of the molecule.

Agammaglobulinemia↗

PubMed1995

A novel point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC.

Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.

Base Sequence↗

PubMed1995

Structural similarity between the pleckstrin homology domain and verotoxin: the problem of measuring and evaluating structural similarity.

An unexpected structural similarity is described between the pleckstrin homology (PH) domain and verotoxin. This similarity has escaped detection primarily due to the differences in topology that exist between the two proteins. By comparing this result with two previously reported similarities for the PH domain, one with the lipocalins and another with the FK506 binding protein, we discuss the problems of measuring and assessing structural similarities.

Amino Acid Sequence↗

PubMed1995

Prediction and analysis of SH2 domain-phosphopeptide interactions.

Src homology 2 (SH2) domains are small protein modules of approximately 100 amino acids that are found in many proteins involved in intracellular signal transduction. They mediate protein-protein interactions and modulate enzyme activity by their ability to bind to specific sequence patterns that contain a phosphorylated tyrosine. As the three-dimensional structures of the phosphatidylinositol (PI) 3-kinase, Lck, Src and Abl SH2 domains have been shown to be similar, we have modelled other SH2 domains that show distinct sequence specificity to allow comparative analysis of SH2-phosphopeptide interactions. The SH2 domains of PLC gamma-Nterm., Nck, Grb2, GAP and Abl have been model-built with high-affinity phosphopeptides fitted into the putative binding sites. For each SH2 domain a detailed analysis of the peptide-protein interaction was performed. It is apparent that specificity is mainly conferred by three to five residues downstream from the phosphotyrosine residue (Y*), especially, although not exclusively, peptide position Y* + 3. The SH2 pocket that binds the Y* + 3 residue is mainly composed of three sections: part of strand beta E going into loop EF, part of alpha B and loop BG. The residues that constitute the Y* + 3 binding pocket show variability that seems to determine which amino acid binds preferentially. Residue position beta E4 seems to play a vital role in the SH2 specificity. This study shows that the development of modelling protocols for SH2 domains whose structure has not been determined can prove very useful in predicting which residues are involved in conferring the affinity and binding specificity of these domains towards distinct phosphotyrosine-containing sequences.

Amino Acid Sequence↗

PubMed1994

Regulation of human type II phosphatidylinositol kinase activity by epidermal growth factor-dependent phosphorylation and receptor association.

Epidermal growth factor (EGF) stimulates phosphatidylinositol PtdIns) hydrolysis in many cell types by effecting the specific interaction between the EGF receptor and phospholipase C gamma. Several studies have suggested that PtdIns 4-kinase activity can also be regulated by EGF, but the mechanism of this stimulation was unclear. We report here that EGF treatment of intact A431 cells increased the association of type II PtdIns kinase with the EGF receptor within 1 min at 37 degrees C. Phosphorylation of immunoprecipitated EGF receptor also increased the association of PtdIns 4-kinase. Furthermore dephosphorylation of phosphoserine residues on the stimulated receptor immune complex led to inactivation of the bound PtdIns 4-kinase, while dephosphorylation of phosphotyrosine residues led to activation. Unlike the stimulated activity measured in total cell and plasma membrane lysates, the changes in activity of the immunoprecipitates were apparent at high substrate concentration. Metabolic labeling was used to show that a 55-kDa phosphoserine and phosphotyrosine-containing protein comigrated with renatured PtdIns 4-kinase activity on SDS-polyacrylamide gel electrophoresis, while in vitro labeling revealed only serine phosphorylation. These data are discussed with reference to the direct regulation of PtdIns 4-kinase by phosphorylation, PtdIns compartmentalization, and the formation of a multienzyme signal transduction complex.

1-Phosphatidylinositol 4-Kinase↗

PubMed1994

Biochemical characterization of the free catalytic p110 alpha and the complexed heterodimeric p110 alpha.p85 alpha forms of the mammalian phosphatidylinositol 3-kinase.

The regulatory (p85 alpha) and catalytic (p110 alpha) subunits of the mammalian phosphatidylinositol 3-kinase have been expressed in insect cells using the baculovirus sytem. The free catalytic subunit p110 alpha and the coexpressed heterodimeric complex of p85 alpha and p110 alpha were purified and their enzymological properties compared. While many kinetic parameters were similar, the coexpressed complex was found to have a 20-fold higher Km for ATP in comparison with the free catalytic subunit p110 alpha using phosphatidylinositol 4,5-bisphosphate as a substrate; no significant difference was detectable when phosphatidylinositol was used. Reconstitution of the p110 alpha.p85 alpha complex in vitro showed that it had the properties of the free p110 alpha and not the p110 alpha.p85 alpha in vivo complex. Therefore, a post-translational modification dependent upon the presence of the regulatory subunit p85 alpha rather than the physical subunit interaction itself is responsible for the observed properties of the lipid kinase activity of the p110 alpha.p85 alpha complex. Phosphatase treatment of the purified lipid kinase complex reduced the high Km for ATP, suggesting that a phosphorylation of the heterodimeric complex (p85 alpha.p110 alpha) caused this effect. This mode of regulation is discussed in the context of lipid kinase activation in vivo.

Animals↗

PubMed1994

Three-dimensional solution structure of the pleckstrin homology domain from dynamin.

BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees. The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99). A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.

Amino Acid Sequence↗

PubMed1994

Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C.

The Kit/stem cell factor receptor (Kit/SCF-R) is a transmembrane tyrosine kinase receptor of importance for the normal development of hemopoietic cells, melanoblasts, and germ cells. We recently reported that protein kinase C (PKC) is involved in a negative feedback loop regulating the Kit/SCF-R by direct phosphorylation on serine residues in the receptor. Inhibition of PKC led to increased SCF-induced tyrosine kinase activity and mitogenicity, but PKC was necessary for SCF-induced motility. In this report we have further examined the modulatory role of PKC on SCF-induced signaling. The ligand-activated Kit/SCF-R associated weakly with GRB2 and induced only little tyrosine phosphorylation of phospholipase C-gamma in porcine aortic endothelial cells transfected with Kit/SCF-R. In contrast, the SCF-stimulated Kit/SCF-R associated efficiently with, and induced tyrosine phosphorylation of, the p85 alpha regulatory subunit of phosphatidyl inositide-3'-kinase (PI-3'-kinase). Both receptor association and tyrosine phosphorylation of p85 alpha were increased after inhibition of PKC, while its serine phosphorylation was decreased. Concomitantly, the specific activity of receptor-associated PI-3'-kinase activity was increased. Inhibition of PI-3'-kinase with wortmannin inhibited SCF-induced mitogenicity. SCF-induced phosphorylation of Raf-1 and activation of ERK2 still occurred after PKC inhibition but was not increased. In conclusion, SCF-induced PI-3'-kinase activation paralleled the increased SCF-induced mitogenicity after inhibition of PKC.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Phosphatidylinositol-3-OH kinase as a direct target of Ras.

Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.

Animals↗

PubMed1994

The catalytic subunit of phosphatidylinositol 3-kinase is a substrate for the activated platelet-derived growth factor receptor, but not for middle-T antigen-pp60c-src complexes.

The interaction of phosphatidylinositol 3-kinase (PI 3-K) with polyoma-virus middle-T antigen-pp60c-src (mT:cSrc) complexes and with the platelet-derived growth factor (PDGF) receptor has been investigated. Firstly, we undertook reconstitution studies, using proteins derived from a baculovirus expression system. The p110 catalytic subunit of the PI 3-K associated with tyrosine kinases only when complexed with the p85 alpha regulatory subunit. Both p85 alpha and p110 were substrates of the PDGF receptor. In contrast, only the p85 alpha subunit was detectably phosphorylated when PI 3-K was associated with mT:cSrc. Secondly, we studied PI 3-K in mammalian cells. In mT-antigen-transformed NIH-3T3 cells neither p85 alpha nor p110 was phosphorylated on tyrosine residues in vivo, even though p85 alpha was a substrate in kinase assays in vitro. In quiescent NIH-3T3 cells, PI 3-K showed detectable activity in vitro; PDGF stimulation resulted in a rapid and transient association of PI 3-K with the receptor, which was correlated with a transient increase in intrinsic P13-K activity (approx. 2-fold). The activated PDGF receptor phosphorylated p110 in vitro, at one major site. In vivo, PDGF stimulation induced tyrosine phosphorylation of p110 that persisted for at least 1 h after stimulation. Immunodepletion of the PDGF receptor from stimulated cell lysates showed that p110 was released from the receptor in a tyrosine-phosphorylated form. From these results we conclude that (i) the mT:cSrc complex and the PDGF receptor differ in their association with PI 3-K activity, (ii) PDGF receptor appears to activate PI 3-K in vivo both by relocation of the enzyme and by stimulation of its intrinsic activity, and (iii) tyrosine phosphorylation of the p110 subunit by the PDGF receptor may play a role in PI 3-K regulation in some circumstances.

3T3 Cells↗

PubMed1994

Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences.

Dynamin, a 100 kDa GTPase, is critical for endocytosis, synaptic transmission and neurogenesis. Endocytosis accompanies receptor processing and plays an essential role in attenuating receptor tyrosine kinase signal transduction. Dynamin has been demonstrated to be involved in the endocytic processing at the cell surface and may play a general role in coupling receptor activation to endocytosis. Src homology (SH) domain dependent protein-protein interactions are important to tyrosine kinase receptor signal transduction. The C-terminus of dynamin contains two clusters of SH3 domain binding proline motifs; these motifs may interact with known SH3 domain proteins during tyrosine kinase receptor activation. We demonstrate here that SH3 domain-containing signal transduction proteins, such as phospholipase C gamma-1 (PLC gamma-1), do indeed bind to dynamin in a growth factor inducible manner. The induction of PLC gamma-1 binding to dynamin occurs within minutes of the addition of platelet derived growth factor (PDGF) to cells. Binding of these signal transduction proteins to dynamin involves specific sorting to individual proline motif clusters and appears to be responsible for co-immunoprecipitation of tyrosine phosphorylated PDGF receptors with dynamin following PDGF stimulation of mammalian cells. The binding of dynamin to SH3 domain-containing proteins may therefore be important for formation of the protein complex required for the endocytic processing of activated tyrosine kinase receptors.

3T3 Cells↗

PubMed1994

An SH3 domain and proline-rich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex.

Neutrophils possess a multicomponent NADPH oxidase system capable of producing large quantities of superoxide in a process known as the respiratory burst (1). Upon stimulation of a phagocytic cell, two cytosolic components of the oxidase, p67phox and p47phox, associate with a membrane-bound flavocytochrome b and a small GTP-binding protein to form a functional enzyme complex. Each of the Phox proteins contains two src homology 3 (SH3) domains, which are of unknown function but are potential mediators of protein-protein interactions between components of the activated oxidase. We have isolated a 47-kDa protein from lysates of differentiated HL60 cells that specifically bound to the carboxyl-terminal SH3 domain of p67phox and not to any other SH3 domain tested. This protein was identified as p47phox, and the putative SH3 domain binding site was located to a carboxyl-terminal proline-rich region. Proline-rich synthetic peptides based on this carboxyl-terminal region specifically inhibited the binding of p47phox to the carboxyl-terminal SH3 domain of p67phox, and sequential truncation defined a unique minimal sequence, which, although similar, does not match the consensus sequence defined for other SH3-binding proteins.

Amino Acid Sequence↗