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M H Cobb

Publications and source records attributed to M H Cobb.

At least 109 records · Page 6Linked to original sources
PubMed1991

Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation.

Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor-activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation. The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.

Amino Acid Sequence↗
PubMed1991

Purification and properties of extracellular signal-regulated kinase 1, an insulin-stimulated microtubule-associated protein 2 kinase.

In rat 1 fibroblasts, insulin has little or no stimulatory effect on the activities of either MAP2 protein kinase or ribosomal protein S6 kinase. In contrast, in rat 1 cells that overexpress the normal human insulin receptor (rat 1 HIRc B; McClain et al. (1987) J. Biol. Chem. 262, 14663-14671), insulin activates both MAP2 and S6 kinase activities close to 5-fold. A MAP2 kinase has been purified from insulin-treated rat 1 HIRc B cells over 6300-fold by chromatography on Q-Sepharose, phenyl-Sepharose, S-Sepharose, phosphocellulose, QAE-Sepharose, UltrogelAcA54, DEAE-cellulose, and a second Q-Sepharose. Its specific activity is approximately 0.8-1 mumol.min-1.mg-1 with MAP2 and 3 mumol.min-1.mg-1 with myelin basic protein. The enzyme preparation contains one major band of Mr = 43,000 upon SDS-polyacrylamide gel electrophoresis, which is immunoblotted by antibodies to phosphotyrosine. A sequence from the 43-kDa band led to the isolation of a cDNA encoding the enzyme, which we have named ERK1 for extracellular signal-regulated kinase (Boulton et al. (1990) Science 249, 64-67).

Amino Acid Sequence↗
PubMed1991

ERKs, extracellular signal-regulated MAP-2 kinases.

A family of protein kinases, known alternatively as microtubule-associated protein-2/myelin basic protein kinases or extracellular signal-regulated kinases, is activated by numerous hormones, growth factors and other extracellular stimuli. At least two members of this family function as intermediate kinases in protein phosphorylation cascades. Their mechanisms of activation may involve autophosphorylation, which occurs on both threonine and tyrosine residues.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1991

Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies.

A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2) and myelin basic protein (MBP) is thought to play a pivotal role in the transduction of signals from many receptors in response to their ligands. A kinase with such activity, named extracellular signal-regulated kinase 1 (ERK1), is activated rapidly by numerous extracellular signals, requires phosphorylation on tyrosine to be fully active, and in vitro can activate a kinase (a ribosomal S6 protein kinase) that is downstream in phosphorylation cascades. From the protein sequence predicted by the rat ERK1 cDNA, peptides were synthesized and used to elicit antibodies. The antibodies recognize both ERK1; a closely related kinase, ERK2; and a third novel ERK-related protein. Using these antibodies we have determined that ERK1 and ERK2 are ubiquitously distributed in rat tissues. Both enzymes are expressed most highly in brain and spinal cord as are their mRNAs. The third ERK protein was found in spinal cord and in testes. The antibodies detect ERKs in cell lines from multiple species, including human, mouse, dog, chicken, and frog, in addition to rat, indicating that the kinases are conserved across species. ERK1 and ERK2 have been separated by chromatography on Mono Q. Stimulation by insulin increases the phosphorylation of both kinases on tyrosine residues, as assessed by immunoblotting with phosphotyrosine antibodies, and retards their elution from Mono Q. Each of these ERKs appears to account for a distinct peak of MBP kinase activity. The activity in each peak is diminished by incubation with either phosphatase 2a or CD45. Therefore, both enzymes have similar modes of regulation and appear to contribute to the growth factor-stimulated MAP2/MBP kinase activity measured in cell extracts.

Amino Acid Sequence↗
PubMed1990

An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control.

A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2), is thought to be an early intermediate in an insulin-stimulated phosphorylation cascade and in a variety of other mammalian cell responses to extracellular signals. A complementary DNA that encodes this protein serine-threonine kinase has been cloned, and the protein designated extracellular signal-regulated kinase 1 (ERK1). ERK1 has striking similarity to two protein kinases, KSS1 and FUS3, from yeast. The yeast kinases function in an antagonistic manner to regulate the cell cycle in response to mating factors. Thus, ERK1 and the two yeast kinases constitute a family of evolutionarily conserved enzymes involved in regulating the response of eukaryotic cells to extracellular signals.

Amino Acid Sequence↗
PubMed1990

Evidence for insulin-dependent activation of S6 and microtubule-associated protein-2 kinases via a human insulin receptor/v-ros hybrid.

The abilities of a series of six mutants of the human insulin receptor, an insulin receptor/v-ros hybrid (IR-ros) and the P68gag-ros transforming protein to stimulate S6 protein kinase have been assessed. Insulin receptor mutants in which either 1 or 2 tyrosine residues have been replaced with phenylalanine (YF1, YF3) have lost some or all of the capacity to mediate the activation of S6 kinase in response to insulin. None of the four mutants that contain deletions (spBam, spBamYF3, iBgl, T-t) elicit an insulin-dependent stimulation of S6 kinase. A previous study of the IRros hybrid receptor demonstrated that it was unable to cause either insulin-stimulated thymidine incorporation or glucose uptake (Ellis, L., Morgan, D. O., Jong, S.-M., Wang, L.-H., Roth, R. A., and Rutter, W. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5101-5105). In contrast, the IRros chimera appears to mediate the activation of S6 protein kinase by insulin. In further evaluating the biological activities of the IRros hybrid, we have examined its effects on a microtubule-associated protein-2 (MAP2) kinase that is thought to be an early target in the cascade of reactions leading to increased S6 phosphorylation (Sturgill, T. W., Ray, L. B., Erickson, E., and Maller, J. L. (1988) Nature 334, 715-718). We find that the IRros receptor stimulates the MAP2 protein kinase from 3- to 6-fold in insulin-treated cells, conferring more than a 30-fold increase in the insulin sensitivity of MAP2 kinase activation.

Animals↗
PubMed1990

Neurotrophic factors, their receptors, and the signal transduction pathways they activate.

Our studies of the spatiotemporal availability of neurotrophic factors, coupled with tagged ligand binding assays that identify cell bearing receptors for these factors, should lead toward defining the physiological roles of these molecules in the animal. The use of the tagged ligands to identify factor-responsive cell lines has also provided new model systems for the examination of ligand-receptor interactions, as well as for the study of the subsequent induction of intracellular response pathways. To obtain insights into such intracellular pathways, we have molecularly cloned genes encoding a family of serine-threonine protein kinases, most closely related to kinases involved in the yeast response to pheromones. These kinases may be crucial regulators of early steps in the response of mammalian cells to neurotrophic factors as well as other extracellular signals.

Amino Acid Sequence↗
PubMed1989

An insulin-stimulated ribosomal protein S6 kinase from rabbit liver.

In this report we describe an activated form of S6 protein kinase in rabbits treated acutely with insulin. The major insulin-stimulated activity in rabbit liver is increased 2- to 5-fold compared to material from untreated animals based on DEAE-cellulose profiles. The activity observed in DEAE-cellulose fractions can be separated into a major and a minor peak, each having very similar chromatographic behavior. Chromatography on DEAE-cellulose, S-Sepharose, heptyl-Sepharose, heparin-agarose, and Mono Q results in greater than 20,000-fold purification of the insulin-stimulated enzyme with a 12% recovery. The stimulated activity has chromatographic properties similar to an S6 protein kinase studied previously in 3T3-L1 cells (Cobb, M. H. (1986) J. Biol. Chem. 261, 12994-12999) and other systems. The enzyme purified from insulin-treated animals contains a major band that migrates in sodium dodecyl sulfate-polyacrylamide gels with Mr congruent to 70,000; this band also appears in the control preparation. Treatment of the insulin-stimulated S6 kinase with the catalytic subunit of phosphatase 2a reduces its activity by 97%. The activity of the inactivated S6 kinase is stimulated nearly 5-fold by a 15-min preincubation with partially purified insulin-stimulated microtubule-associated protein-2 kinase.

Animals↗
PubMed1989

Autophosphorylation activates the soluble cytoplasmic domain of the insulin receptor in an intermolecular reaction.

The cytoplasmic protein-tyrosine kinase domain of the insulin receptor (residues 959-1355) has been expressed as a soluble protein in Sf9 insect cells via a Baculovirus expression vector (Ellis, L., Levitan, A., Cobb, M.H., and Ramos, P. (1988) J. Virol. 62, 1634-1639). The purified protein is a monomer as judged by its behavior in sucrose gradients and on gel filtration in the presence or absence of protamine. The initial rate of autophosphorylation using 3 mM MgCl2 is increased 20-30-fold by protamine. A maximum of 4-5 mol of phosphate are incorporated per mol of enzyme. The activity of the enzyme as a function of phosphorylation state was studied for three substrates: a synthetic dodecapeptide derived from the sequence of the major autophosphorylation site in the insulin receptor, poly(Glu, Tyr), 4:1, and histone 2B. Autophosphorylation of the protein to a stoichiometry of 4-5 mol of phosphate/mol increases its enzymatic activity as much as 200-fold; a 30-fold increase in activity occurs upon addition of 1 mol of phosphate/mol. The activities of unphosphorylated enzyme with the three substrates are 3.4, 2.3, and 0.44 nmol/min/mg, respectively. The activities of the autophosphorylated enzyme with the three substrates are 175, 274, and 45 nmol/min/mg, respectively. Exposure of the autophosphorylated enzyme to ADP results in a loss of phosphate from the enzyme which is associated with a decrease in enzymatic activity. Autophosphorylation of the kinase in the presence or absence of protamine displays a marked dependence on enzyme concentration. Furthermore, the rate of autophosphorylation decreases as the viscosity of the solution increases. Taken together, these data suggest that phosphorylation occurs via an intermolecular reaction.

Adenosine Diphosphate↗
PubMed1988

Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.

Animals↗
PubMed1986

An insulin-stimulated ribosomal protein S6 kinase in 3T3-L1 cells.

A protein kinase that is stimulated from 2-10-fold by insulin and that phosphorylates ribosomal protein S6 has been characterized in 3T3-L1 cells. The detection of this activity in the 100,000 X g supernatant is facilitated by the presence of beta-glycerol phosphate or vanadate in the homogenization buffer. The activity has been purified 55-fold by chromatography on DEAE-cellulose and phosphocellulose. The resulting specific activity is 584 pmol/min/mg of protein. DEAE-cellulose chromatography followed by gel filtration on Ultrogel AcA54 or by glycerol gradient centrifugation suggests that the protein has a molecular mass of 60,000-70,000 daltons. Mg2+, and to a lesser extent Mn2+, will support phosphorylation of S6 by the activity. No proteins tested other than ribosomal protein S6 are phosphorylated. Based on its chromatographic properties and substrate specificity, the enzyme appears to be distinct from several other protein kinases that are known to phosphorylate ribosomal protein S6 in vitro. The complete characterization and purification of this enzyme may be essential to the elucidation of the mechanism of regulation of S6 phosphorylation by insulin.

Animals↗
PubMed1986

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus.

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.

Animals↗
PubMed1986

Insulin-stimulated sodium transport in toad urinary bladder.

Mammalian and teleost insulins increase active sodium transport by the toad urinary bladder at subnanomolar concentrations. This stimulation is evident within 15 min and persists for hours. Porcine proinsulin and a cross-linked derivative of bovine insulin are less effective than porcine insulin in stimulating the short-circuit current (SCC), indicating the specificity appropriate for activation of sodium transport through an insulin receptor. The initial stimulation by insulin of the SCC is not blocked by pretreatment with actinomycin D, puromycin, cycloheximide, or tunicamycin. However, in the presence of any one of these inhibitors the sustained increase in SCC is blocked and the rise is short-lived, lasting only 45 to 90 min. In amphotericin-treated bladders, the addition of insulin did not further stimulate SCC.

Amphotericin B↗
PubMed1985

A comparison of protein synthetic patterns of MDCK cells grown in serum and hormonally defined serum-free medium.

The development of hormonally defined serum-free media (HDM) for the culture of mammalian cells has allowed the study of specific cellular functions in a totally defined environment. Recently, several reports have indicated that there are differences in the basic cellular physiology of cells cultured in HDM when compared to cells cultured by the more traditional method of using serum-supplemented culture media (SM). We report here that there are significant changes in the protein synthetic pattern in MDCK cells grown in HDM. There were no changes exhibited during the first passage in HDM, but following 10 passages in HDM there was an increased isotope ratio of 1) plasma membrane proteins with molecular weights of 12,000, 36,000 and 68,000 and 2) endoplasmic reticulum proteins with molecular weights of 12,000 and 37,000. Additionally, the incorporation of methionine and uridine were significantly increased in cells cultured in HDM for 10 passages. At present, we believe that these changes in the protein synthetic patterns are due at least partially to increased protein synthesis as indicated by monosome/polysome ratios. Therefore, though the use of HDM offers a completely defined system for studying cellular function, results obtained using HDM must be interpreted with caution when comparing them to previous studies that have used SM.

Animals↗
PubMed1985

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors.

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.

Adipose Tissue↗
PubMed1983

Description of a protein kinase derived from insulin-treated 3T3-L1 cells that catalyzes the phosphorylation of ribosomal protein S6 and casein.

Particulate preparations from insulin-treated 3T3-L1 cells retain the enhanced ability to incorporate 32P from [gamma-32P]ATP into ribosomal protein S6 (Smith, C. J., Rubin, C. S., and Rosen, O. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2641-2645). A cyclic AMP-independent protein kinase that phosphorylates S6 and casein and that may be involved in the increase in S6 phosphorylation produced by insulin has been isolated based upon the observation that there is 1.5-3.0-fold higher activity in particulate preparations derived from insulin-treated cells than there is in comparable preparations from control cells. The enzyme activity was purified 2071-fold by KCl extraction, phosphocellulose chromatography, and gel filtration. The S6 phosphorylating activity was also characterized by its behavior on casein-Sepharose and DEAE-cellulose chromatography and its sedimentation in glycerol gradients. None of these procedures resolved the S6 and casein kinase activities. Some of the properties of this kinase, including a molecular weight of about 35,000, inhibition by F- or phosphate, chromatography on DEAE-cellulose and phosphocellulose, and insensitivity to inhibition by GTP, are similar to those of a previously described enzyme, casein kinase I (Dahmus, M. E. (1981) J. Biol. Chem. 256, 3319-3325; Hathaway, G. M., and Traugh, J. A. (1979) J. Biol. Chem. 254, 762-768).

Adipose Tissue↗