Search PubMed⌕ Search

Biomedical subjects

M F White

Publications and source records attributed to M F White.

At least 91 records · Page 5Linked to original sources
PubMed1997

Activation of the phosphatidylinositol 3-kinase serine kinase by IFN-alpha.

During engagement of the type I IFN receptor, IRS-1 is phosphorylated on tyrosine and associates with the p85 regulatory subunit of the phosphatidylinositol (PI) 3'-kinase, which is a dual-specificity enzyme possessing both lipid and serine kinase activities. We sought to determine whether treatment of cells with IFN-alpha activates the PI 3'-kinase serine kinase. 32P-labeling experiments and phosphoaminoacid analysis of immunoprecipitated IRS-1 protein demonstrated that, in addition to tyrosine phosphorylation, IFN-alpha induces its phosphorylation on serine residues. In vitro kinase assays on alphaIRS-1 immunoprecipitates also demonstrated IFN-alpha-dependent serine phosphorylation of IRS-1, suggesting that the protein associates with an IFN-alpha-regulated serine kinase. Furthermore, IFN-alpha-dependent phosphorylation of IRS-1 was detected in in vitro kinase assays on alpha p85 immunoprecipitates, and was inhibited by pretreatment of cells with the specific PI 3'-kinase inhibitor wortmannin, consistent with a regulatory role of the PI 3'-kinase serine kinase on the phosphorylation of the protein. Treatment of cells with wortmannin also inhibited the phosphorylation of the p85 subunit of PI 3'-kinase and the type I IFN-regulated activation of the Map kinase, but had no inhibitory effect on the IFN-alpha-induced activation of Tyk-2 and Jak-1 kinases nor on the activation of Stat-1, Stat-2, and Stat-3. Taken all together, these data establish that the PI 3'-kinase serine kinase is activated by IFN-alpha and may play an important role in the transmission of type I IFN receptor-generated signals.

Androstadienes↗
PubMed1997

The resolving enzyme CCE1 of yeast opens the structure of the four-way DNA junction.

Junction-resolving enzymes exhibit structure-selective binding to DNA, but may also manipulate the DNA structure. CCE1 is a junction-resolving enzyme found in the yeast mitochondrion. To facilitate the analysis of the CCE1-junction interaction, we have exploited the sequence dependence of the cleavage reaction to devise a junction that is refractory to cleavage by this enzyme, even in the presence of magnesium ions. On binding to four-way DNA junctions, pure recombinant CCE1 opens the global structure into a 4-fold symmetrical configuration of arms with an open, chemically reactive centre. The structure of the CCE1-junction complex is independent of the sequence of the junction, and of the presence or absence of magnesium or other ions. This and other functional properties of CCE1 are strikingly similar to those of RuvC resolving enzyme of Escherichia coli.

Base Sequence↗
PubMed1997

The IRS-signalling system during insulin and cytokine action.

The discovery of the first intracellular substrate for insulin, IRS-1, redirected the field of diabetes research and has led to many important advances in our understanding of insulin action. Detailed analysis of IRS-1 demonstrates structure/function relationships for this modular docking molecule, including mechanisms of substrate recognition and signal propagation. Recent work has also identified other structurally similar molecules, including IRS-2, the Drosophila protein, DOS, and the Grb2-binding protein, Gab1, suggesting that this intracellular signalling strategy is conserved evolutionarily and is utilized by an expanding number of receptor systems. In fact, IRS-1 itself has been shown to be important in other growth factor and cytokine signalling systems, including growth hormone and several interleukins. Analysis of mice lacking IRS-1 confirms an important physiological role for this protein in glucose metabolism and general cell growth in the intact animal. Disregulation of the signalling pathways integrated by the IRS proteins may contribute to the pathophysiology of non-insulin-dependent diabetes mellitus or other diseases.

Animals↗
PubMed1997

The insulin signalling system and the IRS proteins.

During the past few years, the insulin signalling system has emerged as a flexible network of interacting proteins. By utilizing the insulin receptor substrate (IRS)-proteins (IRS-1 and IRS-2), the insulin signal can be amplified or attenuated independently of insulin binding and tyrosine kinase activity, providing an extensible mechanism for signal transmission in multiple cellular backgrounds. By employing IRS-proteins to engage various signalling proteins, the insulin receptor avoids the stoichiometric constraints encountered by receptors which directly recruit SH2-proteins to their autophosphorylation sites. Finally, the shared use of IRS-proteins by multiple receptors is likely to reveal important connections between insulin and other hormones and cytokines which were previously unrecognized, or observed but unexplained.

Amino Acid Sequence↗
PubMed1997

Characterization of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe.

The rearrangement and repair of DNA by homologous recombination involves the creation of Holliday junctions, which are cleaved by a class of junction-specific endonucleases to generate recombinant duplex DNA products. Only two cellular junction-resolving enzymes have been identified to date: RuvC in eubacteria and CCE1 from Saccharomyces cerevisiae mitochondria. We have identified a protein from Schizosaccharomyces pombe which has 28% sequence identity to CCE1. The YDC2 protein has been cloned and overexpressed in Escherichia coli, and the purified recombinant protein has been shown to be a Holliday junction-resolving enzyme. YDC2 has a high degree of specificity for the structure of the four-way junction, to which it binds as a dimer. The enzyme exhibits a sequence specificity for junction cleavage that differs from both CCE1 and RuvC, and it cleaves fixed junctions at the point of strand exchange. The conservation of the mechanism of Holliday junction cleavage between two organisms as diverse as S. cerevisiae and S. pombe suggests that there may be a common pathway for mitochondrial homologous recombination in fungi, plants, protists, and possibly higher eukaryotes.

Amino Acid Sequence↗
PubMed1997

Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin.

The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.

Actins↗
PubMed1997

Interaction of wild type and dominant-negative p55PIK regulatory subunit of phosphatidylinositol 3-kinase with insulin-like growth factor-1 signaling proteins.

In a first series of experiments done in the yeast two-hybrid system, we investigated the nature of protein-protein interaction between the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), p55PIK, and several of its potential signaling partners. The region between the Src homology 2 (SH2) domains of p55PIK bound to the NH2 terminus region of p110alpha, as previously shown for p85alpha. Moreover, we found that the insulin-like growth factor-1 receptor (IGF-IR) bound to p55PIK; the interaction occurred at the receptor tyrosine 1316 and involved both p55PIK SH2 domains. Interaction between p55PIK and IGF-IR was seen not only in the yeast two-hybrid system, but also using in vitro binding and coimmunoprecipitation of lysates from IGF-1 stimulated 293 cells overexpressing p55PIK. Further, IGF-I stimulation of these cells led to tyrosine phosphorylation of p55PIK. In 293 cells association of p55PIK with insulin receptor substrate-1 and with IGF-IR was dependent on PI 3-kinase, since it was increased by wortmannin, an inhibitor of PI 3-kinase. Further, by deleting amino acids 203-217 of p55PIK inter-SH2 domain, we engineered a p55PIK mutant unable to bind to the p110alpha catalytic subunit of PI 3-kinase. This mutant had a dominant-negative action on insulin-stimulated glucose transport, since insulin's effect on Glut 4 myc translocation was inhibited in adipocytes expressing mutant p55PIK. Importantly, this dominant-negative mutant was more efficient than wild type p55PIK in associating to IGF-IR and insulin receptor substrate-1 in 293 cells. Taken together, our results show that p55PIK interacts with key elements in the IGF-I signaling pathway, and that these interactions are negatively modulated by PI 3-kinase itself, providing circuitry for regulatory feedback control.

Binding Sites↗
PubMed1997

The IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter for insulin and cytokine action.

Signal transduction by insulin and IGF-1, several interleukins (IL-2, IL-4, IL-9, IL-13), interferons, GH, and other cytokines involves IRS proteins, which link the receptors for these factors to signaling molecules with Src homology-2 domains (SH2-proteins). We recently reported the amino acid sequence of murine IRS-2; in order to examine a potential genetic role for this molecule in disease, we isolated the murine IRS-2 gene and compared the expression pattern of IRS-2 against IRS-1. Like IRS-1, IRS-2 is encoded by a single exon. Whereas IRS-1 is located on murine chromosome 1, IRS-2 is located on murine chromosome 8 near the insulin receptor. IRS-2 is expressed together with IRS-1 in many cells and tissues; however, IRS-2 predominates in murine hematopoietic cells where it may be essential for cytokine signaling; IRS-1 predominates in adipocytes and differentiated 3T3-L1 cells where it contributes to the normal insulin response. In 32D cells, IRS-1 and IRS-2 undergo differential tyrosine phosphorylation during insulin or IL-4 stimulation, as assessed indirectly by interaction with various recombinant SH2 domains. Thus, signaling specificity through the IRS proteins may be accomplished by specific expression patterns and distinct phosphorylation patterns during interaction with various activated receptors.

3T3 Cells↗
PubMed1996

Ca2+ regulates calmodulin binding to IQ motifs in IRS-1.

IRS-proteins couple the receptors for insulin and various cytokines to signalling proteins containing Src homology 2 (SH2) domains. Here we demonstrate that calmodulin, a mediator of Ca(2+)-dependent physiological processes, associates with IRS-1 in a phosphotyrosine-independent manner. IRS-1 coimmunoprecipitated with calmodulin from lysates of Chinese hamster ovary cells expressing IRS-1. The interaction was modulated by Ca2+, and calmodulin binding to IRS-1 was enhanced by increasing intracellular Ca2+ with A23187. In contrast, trifluoperazine, a cell-permeable calmodulin antagonist, decreased binding of calmodulin to IRS-1. Insulin stimulated tyrosine phosphorylation of IRS-1, but did not significantly alter the interaction between calmodulin and IRS-1. IQ-like motifs occur between residues 106-126 and 839-859 of IRS-1. Synthetic peptides based on the these sequences inhibited the association between IRS-1 and calmodulin. These data demonstrate that calmodulin binds to IRS-1 in intact cells in a Ca(2+)-regulated manner, providing a molecular link between the signalling pathways.

Amino Acid Sequence↗
PubMed1996

Insulin-induced egr-1 and c-fos expression in 32D cells requires insulin receptor, Shc, and mitogen-activated protein kinase, but not insulin receptor substrate-1 and phosphatidylinositol 3-kinase activation.

Many studies suggest that insulin utilizes multiple signal transduction pathways. Insulin's effects are initiated by insulin binding to the insulin receptor, resulting in tyrosine phosphorylation of insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, or Shc. We recently demonstrated that immediate-early gene egr-1 transcription was fully induced without phosphorylation of IRS-1 in Chinese hamster ovary cells (Harada, S., Smith, R. M., Smith, J. A., Shah, N. , Hu, D.-Q. & Jarett, L. (1995) J. Biol. Chem. 270, 26632-26638). In the present study, we examined the effects of insulin on immediate-early gene egr-1 and c-fos expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Insulin (17 nM) increased egr-1 and c-fos expression in 32D/IR and 32D/IR+IRS cells, but not in parental cells or 32D/IRS cells, as determined by Northern blot analysis. Insulin treatment (5 min at 37 degrees C) markedly increased tyrosine phosphorylation of several proteins, including the insulin receptor, IRS-1, and Shc, in 32D/IR+IRS cells as determined by immunoprecipitation and Western blot analysis with anti-phosphotyrosine antibody. In contrast, only two tyrosine-phosphorylated proteins, i.e. insulin receptor and Shc, were detected in 32D/IR cells. These data suggest that insulin receptor and Shc phosphorylation is necessary for insulin-induced egr-1 and c-fos expression, but IRS-1 phosphorylation is not necessary or sufficient for the expression of these genes. Furthermore, the effect of specific inhibitors on insulin-induced egr-1 expression was examined. Wortmannin (25 nM), a phosphatidylinositol 3-kinase inhibitor, had no effect on insulin-induced egr-1 expression. In contrast, PD 98059 (30 microM), a mitogen-activated protein kinase kinase inhibitor, totally blocked egr-1 expression induced by insulin. These data indicate that mitogen-activated protein kinase activation, but not phosphatidylinositol 3-kinase activation, is involved in insulin-induced egr-1 expression. Taken together, insulin receptor tyrosine phosphorylation, Shc tyrosine phosphorylation, and mitogen-activated protein kinase activation appear to be the signal transduction pathway responsible for insulin-induced egr-1 expression in 32D cells. These data demonstrate that insulin has multiple signal transduction pathways that vary from cell to cell.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling.

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.

3T3 Cells↗
PubMed1996

Cross-talk between the insulin and angiotensin signaling systems.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

Angiotensin II↗
PubMed1996

The pleckstrin homology domain is the principal link between the insulin receptor and IRS-1.

Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN was proposed to contain another functional PTB domain. Peptide competition experiments demonstrated that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) was essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70(s6k) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions were not required for these insulin actions; however, the IH2(PTB) improved the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increased IRS-1 tyrosine phosphorylation and mediated insulin-stimulated DNA synthesis. The sensitivity of these responses was partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions were deleted together. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction.

Amino Acid Sequence↗
PubMed1996

Tumor necrosis factor (TNF)-alpha inhibits insulin signaling through stimulation of the p55 TNF receptor and activation of sphingomyelinase.

Tumor necrosis factor (TNF)-alpha plays a central role in the state of insulin resistance associated with obesity. It has previously been shown that one important mechanism by which TNF-alpha interferes with insulin signaling is through the serine phosphorylation of insulin receptor substrate-1 (IRS-1), which can then function as an inhibitor of the tyrosine kinase activity of the insulin receptor (IR). However, the receptors and the signaling pathway used by TNF-alpha that mediate the inhibition of IR activity are unknown. We show here that human TNF-alpha, which binds only to the murine p55 TNF receptor (TNFR), is as effective at inhibiting insulin-dependent tyrosine phosphorylation of IR and IRS-1 in adipocytes and myeloid 32D cells as murine TNF-alpha, which binds to both p55 TNFR and p75 TNFR. Likewise, antibodies that are specific agonists for p55 TNFR or p75 TNFR demonstrate that stimulation of p55 TNFR is sufficient to inhibit insulin signaling, though a small effect can also be seen with antibodies to p75 TNFR. Exogenous sphingomyelinase and ceramides, known to be formed by activation of p55 TNFR, inhibit IR and IRS-1 tyrosine phosphorylation and convert IRS-1 into an inhibitor of IR tyrosine kinase in vitro. Myeloid 32D cells expressing IR and IRS-1 are sensitive to this inhibition, but cells expressing IR and IRS-2 are resistant, pointing to an important difference in the biological function between IRS-1 and IRS-2. These data strongly suggest that TNF-alpha inhibits insulin signaling via stimulation of p55 TNFR and sphingomyelinase activity, which results in the production of an inhibitory form of IRS-1.

3T3 Cells↗
PubMed1996

The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

Amino Acid Sequence↗
PubMed1996

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae.

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.

Amino Acid Sequence↗