Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver.

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.     

Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.     

An insulin-sensitive cytosolic protein kinase accounts for the regulation of ATP citrate-lyase phosphorylation.

Purified rat liver ATP citrate-lyase is phosphorylated on serine residues by an insulin-stimulated cytosolic kinase activity partially purified from rat adipocytes [Yu, Khalaf & Czech (1987) J. Biol. Chem. 262, 16677-16685]. The Km for lyase phosphorylation by this hormone-sensitive kinase activity is approx. 3 microM. Two-dimensional tryptic-peptide mapping of the 32P-labelled lyase reveals that the kinase-catalysed phosphorylation occurs primarily on a specific peptide. In intact 32P-labelled adipocytes, insulin enhances the serine phosphorylation of ATP citrate-lyase by 2-3-fold. Tryptic digestion of the 32P-labelled lyase immunopurified from insulin-treated adipocytes also yields one major phosphopeptide. 32P-labelled lyase tryptic peptides derived from labelling experiments in vitro and in vivo exhibit identical electrophoretic and chromatographic migration profiles. Furthermore, radio-sequencing of the phosphopeptide from lyase 32P-labelled in vitro indicates that serine-3 from the N-terminus is phosphorylated by the insulin-stimulated cytosolic kinase, in agreement with previous studies on the position of the phosphoserine residue in ATP citrate-lyase isolated from insulin-treated cells. Taken together, the similarity in site-specific phosphorylation of ATP citrate-lyase from insulin-treated adipocytes to that catalysed by the hormone-activated cytosolic kinase in vitro strongly suggests that this kinase mediates insulin action on lyase phosphorylation in intact cells.     

An insulin-stimulated kemptide kinase purified from rat liver is deactivated by phosphatase 2A.

Insulin action leads to the rapid stimulation of a cytosolic Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) kinase (KIK) that has been recently purified to near homogeneity (Klarlund, J. K., Bradford, A. P., Milla, M. G., and Czech, M. P. (1990) J. Biol. Chem. 265, 227-234). To examine its activation mechanism, purified KIK was treated with purified protein phosphatases. The catalytic subunit of phosphatase 2A inhibited the activity of control KIK by about 50% and abolished the 5-fold elevation in KIK activity due to insulin action. The catalytic subunit of phosphatase 1 with equivalent activity based on dephosphorylation of 32P-labeled phosphorylase alpha had no effect on either control or insulin-stimulated KIK activity. The deactivation of insulin-stimulated KIK by phosphatase 2A was time- and concentration-dependent and was blocked by phosphatase inhibitors. The purified native complexes of phosphatase 2A, phosphatase 2A1, and phosphatase 2A2 similarly deactivated KIK. Analyis of control or insulin-stimulated KIK with two antiphosphotyrosine antibodies by immunoblotting and immunoprecipitation failed to detect the presence of phosphotyrosine in the kinase. These results indicate that KIK is activated by phosphorylation as part of a kinase cascade emanating from insulin receptor stimulation.     

Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection.

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.     

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.     

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.     

Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells

Growth factor activation of serine/threonine protein kinases was studied by treating quiescent Swiss 3T3 cells with epidermal growth factor (EGF) and examining cytosolic extracts for protein kinase activity under conditions inhibitory to calcium- and cyclic nucleotide-dependent kinases. Cytosolic extracts of cells stimulated for 5 min were fractionated by Mono Q fast protein liquid chromatography. Eight peaks of kinase activity were resolved, of which five were stimulated by EGF treatment of cells. These peaks were revealed using the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6 peptide), 40 S ribosomal S6 protein, glycogen synthase, microtubule-associated protein 2, and myelin basic protein as substrates. The peaks varied in the kinetics of their activation by EGF and in their response to insulin. Selected peaks were resolved further by sizing gel chromatography. The results together indicate that at least seven distinct fractions of cytosolic kinase activities are stimulated in Swiss 3T3 cells by EGF. One of these, which phosphorylates both S6 protein and S6 peptide, is similar to the S6 kinase characterized previously in this cell line by others. Four additional activities that also phosphorylate the S6 protein and S6 peptide appear unrelated to this enzyme. Finally, two kinase activities that phosphorylate both myelin basic protein and microtubule associated protein 2 are EGF stimulated. One is similar to an insulin-stimulated microtubule-associated protein 2 kinase described in other cell lines whereas the other seems to represent a novel activity. Several of these EGF-stimulated activities were inactivated by protein phosphatases, suggesting that they might be regulated by phosphorylation.     

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.     

Activation of an insulin-stimulated S6 kinase in 3T3 L1 cell-free extracts by proteolysis

Using chromatography on a Fast S-Sepharose column, the insulin-stimulated S6 kinase can be resolved from other S6 kinases present in 3T3 L1 cell extracts. Only one S6 kinase is greatly activated by insulin (4-5-fold) and phosphorylates S6 with a high stoichiometry (4-5 mol phosphate per mol S6). This insulin-dependent S6 kinase can be activated in cell-free extracts by incubation with high concentrations of Ca2+. This activation is blocked by protease inhibitors such as leupeptin and is mimicked by trypsin. The stimulation does not require the presence of the protein kinase dependent on phospholipids and calcium (PK-C) in the cell extracts. The level of stimulation produced by proteolysis in the cell extracts is comparable to that reached in vivo by incubation with insulin.     

Insulin action rapidly decreases multifunctional protein kinase activity in rat adipose tissue

ATP-citrate lyase in vivo contains three phosphorylation sites on two tryptic peptides (peptides A and B). These phosphorylation sites are under hormonal control. Multifunctional protein kinase (MFPK) from rat liver phosphorylates peptide B on serine and threonine residues whereas cAMP-dependent protein kinase phosphorylates peptide A on a serine residue (Ramakrishna, S., and Benjamin, W. B. (1985) J. Biol. Chem. 260, 12280-12286). We now report that rat adipose tissue MFPK also phosphorylates serine and threonine residues of peptide B of ATP-citrate lyase. When the activity of MFPK was assayed using partially purified (by chromatography on phosphocellulose) cytosol fractions from insulin-treated adipose tissue, it was found that MFPK activity was decreased by over 55%. This decrease in MFPK activity occurs at physiological concentrations of insulin (EC50 = 1 x 10(-10) M). Its onset is rapid and almost maximal at 5 min after the addition of insulin. Even when new protein synthesis is inhibited by cycloheximide, extracts from insulin-treated fat pads have less MFPK activity compared to the control. The insulin effect is maintained after further chromatography on a gel filtration column suggesting that the decrease in MFPK activity is not due to a low molecular weight inhibitor. The insulin-induced decrease in MFPK activity is due to a decrease in Vmax whereas the affinity of this enzyme toward ATP-citrate lyase or ATP is unchanged.     

Evidence for one or more Raf-1 kinase kinase(s) activated by insulin and polypeptide growth factors

The protein product of the Raf-1 proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin and other mitogens. To investigate the mechanism of this activation, we used purified Raf-1 expressed in E. coli as a substrate for a putative Raf-1 protein kinase kinase. In three different insulin-sensitive cell types, insulin activated Raf-1 kinase kinase activity in crude cytosolic cellular fractions. The insulin stimulation of this activity was evident as early as 2 min after exposure to insulin, maximal at 5-8 min, and inapparent at 15 min. Phosphoamino acid analysis of phosphorylated Raf-1 revealed that serine was the primary phosphate acceptor for the insulin-activated kinase or kinases; small amounts of phosphothreonine were also detected. The insulin effect occurred in cells depleted of protein kinase C, and in extracts depleted of endogenous Raf-1 kinase by immunodepletion; these data argue against protein kinase C or Raf-1 kinase itself being the insulin-stimulated activity. The insulin-activated kinase or kinases phosphorylated the Raf-1 protein on multiple sites in vitro, as evidenced by tryptic mapping; at least some of these appeared to overlap with sites phosphorylated in response to serum in intact cells. Several other mitogens and growth factors stimulated Raf-1 kinase kinase activity, including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, serum, and phorbol 12-myristate 13-acetate. This insulin- and mitogen-stimulated Raf-1 kinase kinase activity may play a role in mediating the phosphorylation and possibly the activation of the Raf-1 kinase by insulin and other growth factors.     

An endogenous substrate for the insulin receptor-associated tyrosine kinase

Insulin binding to its receptor stimulates a tyrosine-specific protein kinase. This enzyme phosphorylates the insulin receptor, as well as a variety of exogenous substrates in vitro. In the present studies, we have identified an endogenous substrate for the insulin receptor-associated kinase. We studied insulin-stimulated protein phosphorylation in partially purified insulin receptor preparations from the livers of dexamethasone-treated rats. In this cell-free system, insulin stimulated the phosphorylation of its own receptor as well as of a phosphoprotein of apparent Mr = 120,000 (termed pp120). pp120 was not immunoprecipitated by three anti-receptor antisera, nor was the receptor immunoprecipitated by antisera raised against pp120, suggesting that pp120 is not antigenically related or tightly bound to the insulin receptor. Dose-response curves for receptor and pp120 phosphorylation stimulated by pork insulin were essentially identical, and showed the appropriate specificity (insulin much greater than proinsulin) for a receptor-mediated event. Phosphoamino acid analysis revealed that insulin stimulated the incorporation of 32P predominantly into tyrosine residues of pp120. Casein, an artificial substrate for the insulin receptor kinase, competed with pp120 for insulin-stimulated phosphorylation. Phosphorylation of pp120 was rapid (half-maximal effect within 2 min at 24 degrees C) and, like receptor phosphorylation, was supported with Mn2+ or Mg2+ as divalent cation and ATP as the phosphate donor. While receptor autophosphorylation and artificial substrate phosphorylation were not altered by prior treatment of the rats with dexamethasone, insulin-stimulated pp120 phosphorylation was enhanced in preparations derived from dexamethasone-treated rats, suggesting an alteration of pp120, not the receptor, as a result of dexamethasone-treatment. Further studies of this newly identified endogenous substrate may help clarify the physiologic role of the insulin receptor-associated kinase.     

The human erythrocyte contains two forms of phosphatidylinositol-4-phosphate 5-kinase which are differentially active toward membranes

A human erythrocyte cytosolic phosphatidylinositol-4-phosphate 5-kinase (PIP kinase) and a membrane-bound PIP kinase have been purified by phosphocellulose chromatography. Fractionation of the membrane-bound PIP kinase activities by phosphocellulose separated activity into two peaks, which eluted at 0.6 M NaCl (type I PIP kinase) and 1.0 M NaCl (type II PIP kinase). The cytosolic PIP kinase and the membrane-bound type II PIP kinase are 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable 125I-peptide maps, and are immunochemically indistinguishable, suggesting that they are sequence identical. Antibodies raised to the cytosolic PIP kinase inhibit activity of both the membrane-bound type II and the cytosolic PIP kinases. The type I PIP kinase appears to be distinct from the cytosolic and membrane-bound type II PIP kinase; it is not immunocross-reactive, and antibodies toward type II PIP kinases do not inhibit type I PIP kinase. Further, membrane-bound type II PIP kinase can be removed from type I PIP kinase without loss of activity. Functional characterization of the PIP kinases demonstrates that the type I kinase has a 10-fold lower Km for PIP and a 5-fold higher Km for ATP compared with the type II enzymes. The type I and type II (membrane-bound or cytosolic) PIP kinases are modulated differentially by spermine and heparin. Finally, the type I PIP kinase phosphorylates intrinsic PIP on isolated erythrocyte membranes, whereas the type II PIP kinases have no activity toward native membranes.     

Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, paranitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.Abbreviations EGF Epidermal Growth Factor - IGF I Insulin-like Growth Factor I - PDGF Platelet-Derived Growth Factor - DMEM Dulbecco's Modified Eagle's Medium - FCS Fetal Calf Serum - PBS Phosphate Buffered Saline - PNPP Para-nitrophenylphosphate - BSA Bovine Serum Albumin - -Tyr Antiphosphotyrosine Antibodies - MAP 2 Microtubule-Associated Protein 2 - Hepes N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - EDTA Ethylenediamine Tetraacetic Acid - DTT Dithiothreitol - SDS-PAGE Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis - EGTA [Ethylenebis(oxyethylenenitrilo)] Tetraacetic Acid - TRIS Tris(hydroxymethyl)-Aminoethane - IRSK Insulin Receptor-Associated Serine Kinase - KIK Kemptide Insulin-stimulated Kinase     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin binding and insulin receptor tyrosine kinase activity are not altered in the liver of rats with non-insulin-dependent diabetes

Using the insulin-glucose clamp technique, we have previously shown that an increased sensitivity to insulin in vivo is a characteristic of the liver in rats with non-insulin-dependent diabetes induced by neonatal streptozotocin administration. We have thus studied the properties of liver insulin receptor in that model. 125I-porcine insulin binding was found normal both in isolated plasma membranes and in solubilized, wheat germ agglutinin purified receptors prepared from livers of rats with non-insulin-dependent diabetes, when compared to controls. Basal and insulin-stimulated insulin receptor kinase activities were also found normal for both the autophosphorylation of the beta subunit of the insulin receptor and the phosphorylation of the artificial substrate poly (Glu-Tyr) 4:1. Thus, in that model of chronic insulin deficiency and mild hyperglycemia: 1) liver insulin receptors are not up-regulated; 2) tyrosine kinase activity remains unaffected. This last observation supports the hypothesis that the increased insulin effect in the liver of rats with non-insulin-dependent diabetes is probably distal to the insulin receptor kinase.     

Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-naphthalenyl-methyl) phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa beta-subunit of the insulin receptor (IC50 = 200 microM). The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 microM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa beta-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. Our data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.     

Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this 'in vitro' system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2+ only), treatment at 37 degrees C (1 h), N-ethylmaleimide, phosphate, beta-glycerol phosphate and anti-phosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2+, Ca2+ + calmodulin, Ca2+ + phosphatidylserine + diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2+. This indicates that it is distinct from Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, cyclic AMP- and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase.     

Tyrosine phosphorylation of insulin receptor beta subunit activates the receptor tyrosine kinase in intact H-35 hepatoma cells

The phosphorylation characteristics of insulin receptor from control and insulin-treated rat H-35 hepatoma cells 32P-labeled to equilibrium have been documented. The 32P-labeled insulin receptor is isolated by immunoprecipitation with patient-derived insulin receptor antibodies in the presence of phosphatase and protease inhibitors to preserve the native phosphorylation and structural characteristics of the receptor. The unstimulated insulin receptor contains predominantly [32P] phosphoserine and trace amounts of [32P]phosphothreonine in its beta subunit. In response to insulin, the insulin receptor beta subunit exhibits marked tyrosine phosphorylation and a 2-fold increase in total [32P]phosphoserine contents. High pressure liquid chromatography of the tryptic hydrolysates of the 32P-labeled receptor beta subunit from quiescent cells results in the resolution of up to 9 fractions containing [32P]phosphoserine. The insulin-stimulated tyrosine phosphorylation is concentrated in two of these receptor phosphopeptide fractions, whereas the increase in [32P]phosphoserine content is scattered in low abundance over all receptor tryptic fractions. Insulin receptors affinity-purified by lectin- and insulin-agarose chromatographies from insulin-treated, 32P-labeled cells exhibit a 22-fold increase in the Vmax of receptor tyrosine kinase activity toward histone when compared to controls. The elevated kinase activity of the insulin receptor derived from insulin-treated cells is not due to the presence of hormone bound to the receptor because the receptor kinase activity is assayed while immobilized on insulin-agarose. Furthermore, the insulin-activated receptor kinase activity is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase in vitro. The correlation between the insulin-stimulated site specific tyrosine phosphorylation on receptor beta subunit and the elevation of receptor tyrosine kinase activity strongly suggests that the insulin receptor kinase is activated by hormone-stimulated autophosphorylation on tyrosine residues in intact cells, as previously demonstrated for the purified receptor.