Stimulation of FA and phosphatase-1 activities by insulin in 3T3-L1 cells

The phosphatase-1 activator FA and phosphatase-1 were assayed in 3T3-L1 cells exposed to insulin. The cytosolic FA activity was transiently stimulated (7-8-fold) 1 and 2 min after exposure to 10(-8) M insulin and returned to control values within 5-10 min. Cytosolic phosphatase-1 (assayed after trypsin treatment) was activated (120-140% of controls) between 2 and 5 min and returned to control values within 10 min. Insulin effects were dose-dependent, with maximum stimulation of both activities at 10(-8) M insulin. The possibility that FA and other kinases mediate phosphatase activation by insulin is discussed.     

Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts

We have tested whether growth factors added to serum-deprived BALB/c 3T3 fibroblasts alter the casein kinase II activity measured in cell extracts. A rapid phosphocellulose chromatography method was developed that provides a 40-fold partial purification of casein kinase II activity assayed with the specific substrate peptide Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. Using this technique, kinase activity is stimulated 1.6-2.5-fold when isolated from fibroblasts treated with insulin or insulin-like growth factor I (IGF-I). The activated kinase activity exhibits the specific properties of casein kinase II such as the ability to utilize [gamma-32P]GTP as phosphate donor and marked inhibition by low concentrations of heparin. Activation of casein kinase II appears specific for these hormones because epidermal growth factor and platelet-derived growth factor have no effect on the kinase activity when added to fibroblasts under conditions where they markedly stimulate [3H]thymidine incorporation into DNA. Increases of casein kinase II activity by insulin and IGF-I were detected within 1 min of their addition to cell cultures. IGF-I is more potent in stimulating casein kinase II than insulin in mouse fibroblasts. These results demonstrate that casein kinase II is a selective target for insulin and IGF-I action in BALB/c fibroblasts, consistent with the hypothesis that this kinase plays a role in cellular signaling by these hormones.     

Protein phosphatase-1 and -2A, kinase FA, and casein kinase II in skeletal muscle of streptozotocin diabetic rats.

Protein phosphatase-1 (PP-1) and -2A (PP-2A), two regulatory subunits of PP-1, the glycogen-binding subunit G and inhibitor-2 (I-2), kinase FA, and casein kinase II (CK-II) were investigated in skeletal muscle of diabetic rats 2 days after streptozotocin injection. FA and CK-II activate PP-1 in vitro and might be involved in the activation of PP-1 by insulin. Following muscle fractionation we found that (1) diabetes decreased both basal and trypsin-stimulated PP-1 activities; the decrease was more significant in the glycogen-bound and microsomal fractions than in the cytosol (cytosolic PP-1 decreased as specific activity but not as activity/g of muscle); also PP-2A was lower in diabetic cytosols; (2) less G was immunoprecipitated from diabetic glycogen-bound fractions compared to controls, while I-2 was not significantly changed; (3) diabetes decreased also FA (assayed as PP-1 activator) and CK-II (assayed using a synthetic peptide as substrate); (4) diabetes did not have any effect on phosphorylase (a + b) activity in the glycogen-bound fraction. Altogether the data show that acute diabetes decreased PP-1, one of its regulatory subunits and two potentially physiological regulators of PP-1, in addition to PP-2A. This may indicate that insulin is responsible for the long-term regulation of the same enzymes that are also under acute insulin control.     

Transient effect of platelet-derived growth factor on GLUT4 translocation in 3T3-L1 adipocytes.

We earlier developed a novel method to detect translocation of the glucose transporter (GLUT) directly and simply using c-MYC epitope-tagged GLUT (GLUTMYC). To define the effect of platelet-derived growth factor (PDGF) on glucose transport in 3T3-L1 adipocytes, we investigated the PDGF- and insulin-induced glucose uptake, translocation of glucose transporters, and phosphatidylinositol (PI) 3-kinase activity in 3T3-L1, 3T3-L1GLUT4MYC, and 3T3-L1GLUT1MYC adipocytes. Insulin and PDGF stimulated glucose uptake by 9-10- and 5.5-6.5-fold, respectively, in both 3T3-L1 and 3T3-L1GLUT4MYC adipocytes. Exogenous GLUT4MYC expression led to enhanced PDGF-induced glucose transport. In 3T3-L1GLUT4MYC adipocytes, insulin and PDGF induced an 8- and 5-fold increase in GLUT4MYC translocation, respectively, determined in a cell-surface anti-c-MYC antibody binding assay. This PDGF-induced GLUT4MYC translocation was further demonstrated with fluorescent detection. In contrast, PDGF stimulated a 2-fold increase of GLUT1MYC translocation and 2.5-fold increase of glucose uptake in 3T3-L1GLUT1MYC adipocytes. The PDGF-induced GLUT4MYC translocation, glucose uptake, and PI 3-kinase activity were maximal (100%) at 5-10 min and thereafter rapidly declined to 40, 30, and 12%, respectively, within 60 min, a time when effects of insulin were maximal. Wortmannin (0.1 microM) abolished PDGF-induced GLUT4MYC translocation and glucose uptake in 3T3-L1GLUT4MYC adipocytes. These results suggest that PDGF can transiently trigger the translocation of GLUT4 and stimulate glucose uptake by translocation of both GLUT4 and GLUT1 in a PI 3-kinase-dependent signaling pathway in 3T3-L1 adipocytes.     

Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway

Hormonally stimulated lipolysis occurs by activation of cyclic AMP-dependent protein kinase (PKA) which phosphorylates hormone-sensitive lipase (HSL) and increases adipocyte lipolysis. Evidence suggests that catecholamines not only can activate PKA, but also the mitogen-activated protein kinase pathway and extracellular signal-regulated kinase (ERK). We now demonstrate that two different inhibitors of MEK, the upstream activator of ERK, block catecholamine- and beta(3)-stimulated lipolysis by approximately 30%. Furthermore, treatment of adipocytes with dioctanoylglycerol, which activates ERK, increases lipolysis, although MEK inhibitors decrease dioctanoylglycerol-stimulated activation of lipolysis. Using a tamoxifen regulatable Raf system expressed in 3T3-L1 preadipocytes, exposure to tamoxifen causes a 14-fold activation of ERK within 15-30 min and results in approximately 2-fold increase in HSL activity. In addition, when differentiated 3T3-L1 cells expressing the regulatable Raf were exposed to tamoxifen, a 2-fold increase in lipolysis is observed. HSL is a substrate of activated ERK and site-directed mutagenesis of putative ERK consensus phosphorylation sites in HSL identified Ser(600) as the site phosphorylated by active ERK. When S600A HSL was expressed in 3T3-L1 cells expressing the regulatable Raf, tamoxifen treatment fails to increase its activity. Thus, activation of the ERK pathway appears to be able to regulate adipocyte lipolysis by phosphorylating HSL on Ser(600) and increasing the activity of HSL.     

Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.     

Fatty acid infusion selectively impairs insulin action on Akt1 and protein kinase C lambda /zeta but not on glycogen synthase kinase-3

To determine the mechanism(s) for insulin resistance induced by fatty acids, we measured the ability of insulin to activate phosphoinositide 3-kinase (PI3K) and multiple distal pathways in rats. Following a 5-h infusion of lipid or glycerol (control), rats underwent a euglycemic hyperinsulinemic clamp. Insulin stimulated IRS-1-associated PI3K activity in muscle of glycerol-infused rats 2.4-fold but had no effect in lipid-infused rats. IRS-2- and phosphotyrosine-associated PI3K activity were increased 3.5- and 4.8-fold, respectively, by insulin in glycerol-infused rats but only 1.6- and 2.3-fold in lipid-infused rats. Insulin increased Akt1 activity 3.9-fold in glycerol-infused rats, and this was impaired 41% in lipid-infused rats. Insulin action on Akt2 and p70S6K were not impaired, whereas activation of protein kinase C lambda/zeta activity was reduced 47%. Insulin inhibited glycogen synthase kinase 3alpha (GSK-3alpha) activity by 30% and GSK-3beta activity by approximately 65% and increased protein phosphatase-1 activity by 40-47% in both glycerol- and lipid-infused rats. Insulin stimulated glycogen synthase activity 2.0-fold in glycerol-infused rats but only 1.4-fold in lipid-infused rats. Thus, 1) elevation of fatty acids differentially affects insulin action on pathways distal to PI3K, impairing activation of Akt1 and protein kinase C lambda/zeta and 2) insulin action on glycogen synthase can be regulated independent of effects on GSK-3 and protein phosphatase-1 activity in vivo.     

Identification of the sites on rabbit skeletal muscle protein phosphatase inhibitor-2 phosphorylated by casein kinase-II

Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C-terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2.     

An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells

Insulin stimulates the phosphorylation of the 40 S ribosomal subunit protein, S6, in intact 32P-labeled H4IIE-C3 cells, a rat hepatoma line. Cell-free cytosolic extracts from H4 cells exhibit a 5- to 10-fold increase in S6 protein kinase activity (measured by transfer of 32P to exogenous 40 S rat liver ribosomal subunits) when prepared from cells exposed to insulin prior to homogenization. Stimulation of S6 phosphorylation in intact cells and activation of S6 protein kinase in cell-free extracts are both detectable within 2 min after insulin, and are maximally stimulated by 10 min. Half-maximal stimulation is observed at 10(-11) M insulin. The stimulated S6 kinase activity requires ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to be present during the kinase assay for full expression. Despite the presence of a 5- to 10-fold increase in S6 protein kinase activity, the extracts from insulin-treated cells exhibit no stimulated kinase activity toward casein, histone, or ATP-citrate lyase assayed under the conditions employed for S6. Thus, insulin mediates the rapid activation of protein kinase specific for ribosomal protein S6 by an as yet unidentified mechanism.     

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.     

Evidence that erythroid-type glucose transporter intrinsic activity is modulated by cadmium treatment of mouse 3T3-L1 cells

Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.     

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.     

Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.     

A Capillary Electrophoresis-Based Assay for Protein Kinases and Protein Phosphatases Using Peptide Substrates

A novel procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the R_II subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.     

Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.     

Effect of pH on binding of agonists and antagonists to rat heart muscarinic receptors.

Insulin, epidermal growth factor (EGF), platelet-derived growth factor, multiplication-stimulating activity and 10% foetal-calf serum each stimulated the phosphorylation of a cytosolic Mr-22000 acidic heat-stable protein in Swiss mouse 3T3-L1 adipocytes. Phosphorylation of this protein was not stimulated by isoprenaline or dibutyryl cyclic AMP. The effect of insulin was maximal (3-fold increase) by 10 min; half-maximal stimulation was observed at 70 pM-insulin. Both [32P]phosphoserine and [32P]phosphothreonine residues were present in the Mr-22000 protein after insulin- and growth-factor-stimulated phosphorylation, but no [32P]phosphotyrosine. The major site of insulin- and EGF-stimulated phosphorylation appeared to be a threonine residue, in contrast with previously studied insulin-stimulated phosphorylation of serine residues. Insulin treatment appeared to result in a shift of the protein toward the anode on isoelectric focusing. Insulin and EGF present simultaneously did not lead to phosphorylation beyond that seen with each hormone singly. We surmise that insulin, EGF and perhaps other growth factors may activate a common protein kinase or inhibit a common protein phosphatase in 3T3-L1 adipocytes which acts on the Mr-22000 protein.     

Insulin-stimulated microtubule associated protein kinase is detectable by analytical gel chromatography as a 35-kDa protein in myocytes, adipocytes, and hepatocytes

Insulin stimulates a novel Ser/Thr kinase, which phosphorylates microtubule associated protein-2 (MAP-2) in vitro. MAP kinase was studied in cell models of the principal insulin responsive tissues using analytical fast-protein liquid chromatography for partial purification of the enzyme. Stimulation of MAP kinase (1.3- to 2-fold) by insulin was readily detected in BC3H1 smooth and 23A2 skeletal muscle cells; 3T3-L1 adipocytes; and isolated rat hepatocytes and adipocytes. No phosphatase activity was detectable under the assay conditions used, proving that stimulation of a kinase, not inhibition of a phosphatase, is responsible for the increased incorporation of 32PO4 catalyzed by supernatants from insulin-treated 3T3-L1 cells. In H4 hepatoma cells, stimulation of MAP kinase was much less evident after gel filtration in comparison to the other cell types. The activated enzyme present in supernatants from insulin-treated cells migrated as a single peak of approximately 35 kDa apparent molecular mass (except in the case of isolated hepatocytes in which a shoulder was present). These results suggest that the insulin-stimulatable MAP kinase may be ubiquitous in insulin responsive cells.     

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.