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R A Kohanski

Publications and source records attributed to R A Kohanski.

At least 37 records · Page 2Linked to original sources
PubMed1997

A tyrosine kinase assay using reverse-phase high-performance liquid chromatography.

Reverse-phase HPLC can be used as a very precise and accurate routine assay for peptide phosphorylation by protein kinases that has advantages over other methods. In particular, peptides with native amino acid sequences can be used without the need for radioisotopes. However, reaction conditions that are employed can often present difficulties in recovery and quantitation of phospho- and apo-peptides. Two general problems were encountered; First, variation in the retention times of peptides and an increasing width of the injection front which can interfere with quantitation both resulted from repeated sample injections. These were caused mostly by the presence of carrier bovine serum albumin used to reduce loss of peptides during the reaction and by high concentrations of ATP used to study the kinetics of enzyme catalyzed reactions. These problems were solved by regular washing of the reverse-phase column, thus allowing a broad range of peptide and ATP concentrations to be used. Second, the stability of peptides used in the assay was affected by dithiothreitol in combination with manganese. The former is a common reagent of kinase purifications and the latter is often the metal cofactor used in kinase reactions. Minimizing the concentration of dithiothreitol or using magnesium resolved these difficulties. Consideration of these factors is therefore important when using reverse-phase HPLC to monitor peptide phosphorylation in protein tyrosine kinase assays.

Adenosine Triphosphate↗
PubMed1997

Stat5 is a physiological substrate of the insulin receptor.

Using the cytoplasmic domain of the insulin receptor (IR) in a yeast two-hybrid screen, we identified a cDNA clone encoding the C-terminal 308 amino acids of human Stat5b (Stat5b-Ct). Stat5b-Ct is tyrosine phosphorylated by purified IR kinase domain in vitro. Insulin stimulates tyrosine phosphorylation of overexpressed Stat5b-Ct and endogenous Stat5 in cells overexpressing IR. Stat5 may be a direct target of the IR and, as a member of the Stat family of transcription factors, may play a role in the regulation of gene transcription by insulin. In support of this hypothesis, perfusion of mouse liver with insulin promotes rapid tyrosine phosphorylation of Stat5 and activation of Stat5 DNA binding. Moreover, refeeding of fasted mice leads to rapid tyrosine phosphorylation and stimulation of enhanced DNA-binding activity of Stat5 extracted from liver, skeletal muscle, and adipose tissues. Taken together, our data strongly suggest that IR interacts with and phosphorylates Stat5 in vitro and in tissues physiologically sensitive to insulin.

3T3 Cells↗
PubMed1996

Purification, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involved in the activation of POX1.

Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the beta-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480-24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.

Acyl-CoA Oxidase↗
PubMed1995

Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase.

The unusually large (approximately 600 to > 3000 kDa) myosin-associated proteins of the titin/twitchin superfamily are considered to be important cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60-240 modular fibronectin-type-III and immunoglobulin-C2 repeats in these proteins which further contain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitchin was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19-kDa myosin regulatory light chains. The twitchin kinase phosphorylated purified light chains on Thr15 in a region which shared a high degree of similarity with the phosphorylation site for vertebrate smooth muscle myosin light chain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the first potential substrate for any of the giant protein kinases and support a dual role of twitchin in molluscan muscle as a cytoskeletal protein as well as a myosin light chain kinase.

Amino Acid Sequence↗
PubMed1995

Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.

Base Sequence↗
PubMed1995

Tyrosine dephosphorylation of nuclear proteins mimics transforming growth factor beta 1 stimulation of alpha 2(I) collagen gene expression.

Transforming growth factor beta 1 (TGF-beta 1) exerts a positive effect on the transcription of genes coding for several extracellular matrix-related products, including collagen I. We have previously identified a strong TGF-beta 1-responsive element (TbRE) in the upstream promoter sequence of the alpha 2(I) collagen (COL1A2) gene. Our experiments have shown that TGF-beta 1 stimulates COL1A2 transcription by increasing binding of an Sp1-containing complex (TbRC) to the TbRE. They have also suggested that the change occurs via posttranslational modification of a protein(s) directly or indirectly interacting with Sp1. Here, we provide evidence showing that tyrosine dephosphorylation of nuclear proteins mimics the stimulation of COL1A2 transcription by the TGF-beta 1-activated signaling pathway. Preincubation of nuclear extracts with protein tyrosine phosphatase (PTPase) but not with protein phosphatase type 2A (PP2A), a serine/threonine phosphatase, enhanced binding of the TbRC to the same degree as culturing cells in TGF-beta 1. Consistent with these in vitro findings, genistein, a tyrosine kinase inhibitor, led to markedly increased COL1A2 gene expression, whereas sodium orthovanadate, a tyrosine phosphatase inhibitor, decreased it substantially. These results were supported by transfection experiments showing that genistein and sodium orthovanadate have opposite effects on TbRE-mediated transcription. Moreover, nuclear proteins isolated from genistein-treated cells were found to interact with the TbRE significantly more than those from untreated cells. Furthermore, pretreatment of cells with sodium orthovanadate virtually abrogated nuclear protein binding to the TbRE, but not to a neighboring cis-acting element unresponsive to TGF-beta 1. The results of this study, therefore, provide the first correlation between tyrosine dephosphorylation, increased binding of a transcriptional complex, and TGF-beta 1 stimulation of gene expression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Absence of insulin receptor gene mutations in three insulin-resistant women with the polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) are markedly insulin-resistant, but the molecular mechanisms of these changes and their relationship to the hyperandrogenic state remain to be clarified. Mutations have recently been identified in the insulin receptor gene of patients with extreme forms of insulin resistance associated with hyperandrogenism (eg, type A insulin resistance), and these mutations account for the insulin resistance in such patients. We performed this study to determine whether mutations in the coding portion of the insulin receptor gene were responsible for insulin resistance in PCOS. Insulin binding studies using cultured skin fibroblasts of three obese (body mass index > 27 kg/m2) women with PCOS (ie, mild hyperandrogenemia and chronic anovulation of unknown etiology) and documented insulin resistance showed no apparent abnormalities in either the number or affinity of insulin binding sites. Direct sequencing of all 22 exons of the insulin receptor gene from two of the women with PCOS did not reveal any mutations. Furthermore, both alleles of the gene were expressed at equal levels. In a third insulin-resistant PCOS woman, there was no evidence for a mutation in the coding portion of the insulin receptor gene as determined by denaturing gradient gel electrophoresis (DGGE). We conclude that the insulin resistance in these PCOS women was caused by a defect extrinsic to the insulin receptor.

Adult↗
PubMed1993

Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain.

Kinetic analysis of autophosphorylation was done using a non-Michaelis-Menten kinetic model. This model describes autophosphorylation in terms of a fast reaction phase, a slow reaction phase, and a partition function for the two phases. Kinetic parameters determined by this new approach show that insulin stimulates autophosphorylation by promoting (1) a 10-fold increase in the rate constant for the fast phase of the reaction and (2) a 2-fold increase in the partition function favoring the fast phase. Insulin did not significantly affect the binding constant for ATP in this fast phase. Kinetic parameters obtained for the cytoplasmic kinase domain were similar to those obtained for the native insulin receptor in the absence of insulin. The insulin receptor has three subdomains encompassing its seven autophosphorylation sites. The juxtamembrane sites react primarily in the slow kinetic phase, favored by the absence of stimulation and low ATP concentrations. The carboxy-terminal and central autophosphorylation subdomains react primarily in the fast kinetic phase, favored by raising the ATP concentration and/or the presence of insulin. These observations demonstrate that: (1) both ATP and insulin regulate reaction in each autophosphorylation subdomain, (2) insulin stimulation occurs predominantly in the central and carboxy-terminal regions, and (3) autophosphorylation observed with the cytoplasmic kinase domain was similar to native insulin receptor in the absence of insulin. These findings are consistent with conclusions based on the kinetic analysis of autophosphorylation.

3T3 Cells↗
PubMed1993

Insulin receptor autophosphorylation. II. Determination of autophosphorylation sites by chemical sequence analysis and identification of the juxtamembrane sites.

Autophosphorylation sites of the human insulin receptor were identified by microsequence analysis of 19 discrete tryptic [32P]phosphopeptides, purified from the autophosphorylated cytoplasmic kinase domain (CKD). Seventeen phosphopeptides were generated by cleavage at Arg and/or Lys, but two phosphopeptides were generated reproducibly by anomalous cleavages. Two new sites were identified in the juxtamembrane region of the intact insulin receptor beta-subunit (the amino terminus of the CKD), including phosphotyrosines 965 and 972. Three sites in the central region, including phosphotyrosines 1158, 1162, and 1163, were identified from six phosphopeptides; tyrosine at 1158 was the least phosphorylated. Monophosphopeptides contained phosphotyrosine at either residue 1158 or 1163, but not at 1162. Bisphosphorylation included phosphotyrosines only at 1162 and 1163. The two autophosphorylation sites near the carboxy terminus were found in seven phosphopeptides, including phosphotyrosines at 1328 and 1334. When mapped by reverse-phase high-performance liquid chromatography, these phosphopeptides eluted in the order central domain, first; carboxy-terminal region, second; and juxtamembrane (amino-terminal) domain, last.

Adenosine Triphosphate↗
PubMed1991

Control of insulin receptor autophosphorylation by polypeptide substrates: inhibition and stimulation by interaction with the catalytic subunit.

Autophosphorylation of purified insulin receptor, in the absence of insulin, was stimulated by selected polypeptide substrates. In the presence of 1 microM insulin these peptides inhibited autophosphorylation. Stimulation was observed with reduced [S-(carboxamidomethyl)cysteinyl]lysozyme (RCAM-lysozyme) and three peptides generated by CNBr cleavage, V8 proteinase digestion and/or chemical modification. We also generated two peptide substrates from RCAM-lysozyme which did not stimulate receptor autophosphorylation and were very weak inhibitors. As a control peptide, the simple substrate angiotensin inhibited receptor autophosphorylation in the absence or presence of insulin. However, stimulatory peptide, but not insulin, significantly shifted the concentration dependence for inhibition by angiotensin. The stimulatory peptides also increased autophosphorylation of the cloned cytoplasmic domain of the kinase [R-BIRK; Villalba, M., Wente, S. R., Russell, D. S., Ahn, J., Reichelderfer, C. F., & Rosen, O. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7848]. Therefore, stimulation occurs by interaction with the cytoplasmic process of the beta-subunit and not through interaction with the insulin binding alpha-subunit of the native receptor. Autophosphorylation was analyzed by mapping 32P-labeled tryptic phosphopeptides from the beta-subunit and from R-BIRK. Nearly identical phosphopeptide maps were found, comparing first, basal R-BIRK and basal native receptor, second, peptide- and insulin-stimulated native receptor, and third, peptide-stimulated R-BIRK and insulin-stimulated native receptor. Therefore, R-BIRK functions as a basal-state enzyme and can be stimulated in an insulin-like manner. On the basis of these observations, stimulation by insulin and by peptides yields similar functional results, but by apparently different mechanisms.

Adenosine Triphosphate↗
PubMed1991

The native alpha 2 beta 2 tetramer is the only subunit structure of the insulin receptor in intact cells and purified receptor preparations.

The native subunit structure of the insulin receptor was reinvestigated by two-dimensional nonreducing/reducing gel electrophoresis. Human insulin receptor expressed in murine fibroblasts was found to be a single oligomer, the alpha 2 beta 2 heterotetramer. The structure was assessed using receptor metabolically labeled with [35S]methionine, and using receptor autophosphorylation at two levels of purification: the insulin affinity-purified receptor and the more commonly used wheat germ agglutinin-Sepharose-enriched fraction from whole membrane extracts. Lower molecular weight oligomers and free subunits were observed only upon heating the sample prior to electrophoresis. This artifact of sample handling was dependent upon three factors: (i) temperature, (ii) time of heating, and (iii) impurities typically present in partially purified receptor preparations. We conclude that the alpha 2 beta 2 tetramer is the only insulin receptor subunit structure native in intact cells and subsequently isolated from cell membranes.

Animals↗
PubMed1990

Primary structure of avian hepatic rhodanese.

Rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1.) was purified from chicken livers and its amino acid sequence was determined. The enzyme has a specific activity of 676 IU and a molecular weight of 32,255. The primary structure of 289 amino acids was solved by sequential Edman degradation of overlapping peptides obtained by selected enzymatic and chemical cleavages. The amino terminus was blocked, and the carboxy-terminus was heterogeneous. Comparison of the primary structure with bovine liver rhodanese showed 212 identically matched amino acids, and the majority of amino acid differences were conservative substitutions. Reaction of the enzyme with a 1.4-fold molar excess of [2-14C]iodoacetate led to inactivation of the enzyme and carboxymethylation of Cys-244; this modification was blocked by the substrate thiosulfate.

Amino Acid Sequence↗
PubMed1989

Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids.

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the [32P]phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.

Adenosine Triphosphate↗
PubMed1988

Conformational states of the insulin receptor.

Insulin binding to the alpha-subunit of the purified insulin receptor changed the interaction between beta-subunits. This conformational change was demonstrated after labeling the receptor's beta-subunit by autophosphorylation in the absence of insulin, and then crosslinking the subunits to each other with bis (sulfosuccinimidyl) suberate. The convalent oligomers were resolved by reduction and denaturing gel electrophoresis. Insulin increased the rate of crosslinking, especially the formation of beta-beta dimers. These results support a conformational change following insulin binding, and may reflect the insulin-induced activation of autophosphorylation.

Animals↗
PubMed1987

Effect of phenylarsine oxide on insulin-dependent protein phosphorylation and glucose transport in 3T3-L1 adipocytes.

We have reported previously that phenylarsine oxide (PAO) blocks insulin-stimulated glucose transport in 3T3-L1 adipocytes (Frost, S. C., and Lane, M. D. (1985) J. Biol. Chem. 260, 2646-2652). As shown in the present study, the locus of inhibition is post-receptor. Insulin stimulated the extent of receptor autophosphorylation in solution and in the intact cell by approximately 4-fold. PAO had no effect on this activity. Using reduced and carboxamidomethylated lysozyme as a substrate for the tyrosine-specific receptor, insulin stimulated the rate of receptor kinase-catalyzed substrate phosphorylation by 2-fold; PAO had no effect on this stimulation. However, the insulin-stimulated, serine-specific phosphorylation of two endogenous phosphoproteins (pp24 and pp240) in the intact cell was blocked by 25 microM PAO. These complementary in situ and in vitro studies demonstrate that the inhibition by PAO must be distal to the insulin receptor's protein tyrosine kinase activity.

Adipose Tissue↗
PubMed1986

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes.

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.

Adipose Tissue↗
PubMed1986

Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes.

The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.

Adipose Tissue↗