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.     

Purification of a novel insulin-stimulated protein kinase from rat liver

We previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.     

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte

We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.     

Role of protein phosphorylation in the maturation-induced activation of a myelin basic protein kinase from sea star oocytes

We have previously described the purification of a myelin basis protein (MBP) kinase from maturing sea star oocytes (Sanghera, J. S., Paddon, H. B., Bader, S. A., and Pelech, S. L. (1990) J. Biol. Chem. 265, 52-57). The ability of the purified 44-kDa protein to bind azido-ATP and undergo autophosphorylation on the serine residue implied that it is a protein kinase. Furthermore, partial amino acid sequence data has revealed that it is a novel protein kinase, which we have provisionally designated p44mpk. Autophosphorylation of p44mpk to 0.7 mol of phosphate/mol of enzyme was correlated with a modest (approximately 17%) increase in the MBP-phosphorylating activity of the kinase. Rabbit polyclonal antibody raised against purified p44mpk recognized on immunoblots the protein in highly purified preparations as well as crude oocyte extracts. The affinity-purified anti-p44mpk antibody could immunoprecipitate active kinase, but a subpopulation of the antibody also appeared to be inhibitory. Using this antibody, we have demonstrated that the up to 12-fold stimulation of the cytosolic MBP-phosphorylating activity of this kinase that occurs during sea star oocyte maturation is not due to an increase in the amount of enzyme protein, either from a redistribution within the oocyte or protein synthesis. A slight retardation of the migration of the activated p44mpk on sodium dodecyl sulfate-polyacrylamide gels and its tighter interaction with a MonoQ column is consistent with phosphorylation of the kinase during maturation. p44mpk underwent enhanced phosphorylation when oocytes prelabeled with [32P]orthophosphate were induced to mature with 1-methyladenine. The stimulated MBP-phosphorylating activity of p44mpk in cytosols from maturing oocytes was partly stabilized by the presence of the phosphatase inhibitor beta-glycerol phosphate. Furthermore, treatment of purified p44mpk with protein phosphatase 2A and alkaline phosphatase resulted in 56 and 86% decreases, respectively, in the activity of the kinase. Together, these findings strongly implicate a role for phosphorylation of p44mpk in its activation during sea star oocyte maturation.     

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.     

A purified S6 kinase kinase from Xenopus eggs activates S6 kinase II and autophosphorylates on serine, threonine, and tyrosine residues.

S6 kinases I and II have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 kinase clone, closely related to S6 kinase II, was subsequently identified and the protein product was expressed in a baculovirus system. Using this protein, termed "rsk" for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine kinase termed rsk kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk kinase, whereas alpha-casein, histone H1, protamine, and phosvitin are not. rsk kinase is inhibited by low concentrations of heparin as well as by beta-glycerophosphate and calcium. Activation of rsk kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. However, in vitro, rsk kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, identifying it as a "dual specificity" enzyme. Purified rsk kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serine/threonine protein phosphatase 2A. Phosphatase-treated S6KII can be reactivated by rsk kinase, and S6 kinase activity in resting oocyte extracts increases significantly when purified rsk kinase is added. The availability of purified rsk kinase will enhance study of the signal transduction pathway(s) regulating phosphorylation of ribosomal protein S6 in Xenopus oocytes.     

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.     

Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal

Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.     

Purification and properties of a protamine kinase from bovine kidney microsomes.

About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.     

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.     

A comparative study of the microsomal S6 phosphatase and phosphorylase phosphatase activities in rat liver.

Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations.     

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.     

Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro

A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.     

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.     

Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor

The effect of 8-bromo-cAMP and forskolin on the phosphorylation state and protein kinase activity of the insulin receptor was evaluated in cultured IM-9 lymphoblasts. 8-Bromo-cAMP (1 mM) or forskolin (10 microM) enhanced the phosphorylation of the insulin receptor purified from 32P-labeled cells by affinity chromatography on wheat germ agglutinin-agarose and immunoprecipitation with monoclonal antibody. In the absence of insulin, phosphorylation of the beta subunit of the receptor was increased approximately 2-fold by raising intracellular cAMP. Phosphoamino acid analysis of the beta subunit following treatment of cells with forskolin revealed an increase in phosphoserine and phosphothreonine residues. In contrast, the insulin-stimulated phosphorylation of the receptor occurred on serine, threonine, and tyrosine residues and was diminished by prior exposure of cells to forskolin. Pulse-chase experiments indicated that forskolin did not enhance the turnover of phosphate on the receptor of cells previously exposed to insulin. Furthermore, extracts from forskolin-treated cells did not differ from control extracts in their capacity to dephosphorylate 32P-labeled receptor isolated from cells treated with insulin. The insulin-dependent tyrosine protein kinase activity of the receptor isolated from forskolin-treated cells was approximately 50% as active as the receptor isolated from either control or insulin-treated cells. This was assessed using both histone and a peptide synthesized in accordance with the deduced amino acid sequence of a potential autophosphorylation site of the human receptor (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys) as substrates for the protein kinase reaction. These results suggest that agents that raise intracellular cAMP increase phosphorylation of the insulin receptor on serine and threonine residues, reduce insulin-mediated receptor phosphorylation on tyrosine, serine, and threonine residues, and inhibit the insulin-dependent tyrosine protein kinase activity of the receptor. Thus cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.     

Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases.

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.     

In vivo phosphorylation of insulin receptor substrate 1 at serine 789 by a novel serine kinase in insulin-resistant rodents

Insulin resistance is a key pathophysiologic feature of obesity and type 2 diabetes and is associated with other human diseases, including atherosclerosis, hypertension, hyperlipidemia, and polycystic ovarian disease. Yet, the specific cellular defects that cause insulin resistance are not precisely known. Insulin receptor substrate (IRS) proteins are important signaling molecules that mediate insulin action in insulin-sensitive cells. Recently, serine phosphorylation of IRS proteins has been implicated in attenuating insulin signaling and is thought to be a potential mechanism for insulin resistance. However, in vivo increased serine phosphorylation of IRS proteins in insulin-resistant animal models has not been reported before. In the present study, we have confirmed previous findings in both JCR:LA-cp and Zucker fatty rats, two genetically unrelated insulin-resistant rodent models, that an enhanced serine kinase activity in liver is associated with insulin resistance. The enhanced serine kinase specifically phosphorylates the conserved Ser(789) residue in IRS-1, which is in a sequence motif separate from the ones for MAPK, c-Jun N-terminal kinase, glycogen-synthase kinase 3 (GSK-3), Akt, phosphatidylinositol 3'-kinase, or casein kinase. It is similar to the phosphorylation motif for AMP-activated protein kinase, but the serine kinase in the insulin-resistant animals was shown not to be an AMP-activated protein kinase, suggesting a potential novel serine kinase. Using a specific antibody against Ser(P)(789) peptide of IRS-1, we then demonstrated for the first time a striking increase of Ser(789)-phosphorylated IRS-1 in livers of insulin-resistant rodent models, indicating enhanced serine kinase activity in vivo. Taken together, these data strongly suggest that unknown serine kinase activity and Ser(789) phosphorylation of IRS-1 may play an important role in attenuating insulin signaling in insulin-resistant animal models.     

Characterization of an activated ribosomal S6 kinase variant from maturing sea star oocytes: association with phosphatase 2A and substrate specificity.

Two ribosomal protein S6 kinases (i.e., pp52(S6K) and pp70(S6K)) of the p70 S6 kinase family were markedly activated during meiotic maturation of Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52(S6K) by approximately 50,000-fold with a specific enzyme activity of 1.6 micromol per min per mg. The purified enzyme apparently featured the N- and C-terminal regions of pp70(S6K) as it immunoreacted with antibodies directed to peptides patterned after these amino acid sequences in mammalian pp70(S6K). pp52(S6K) was inhibited by fluoride (IC(50) approximately 60 mM), but was relatively insensitive to beta-glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn(2+), Zn(2+), and Ca(2+). The consensus sequence for substrate phosphorylation was determined to be RXXSXR, which was partially distinct from mammalian p70(S6K) in its requirement for an amino-terminal arginine. Phosphorylation of ribosomal protein S6 by p52(S6K) occurred exclusively on serine on at least five tryptic peptides. Inhibition of sea star p52(S6K) phosphotransferase activity after treatment with protein serine/threonine phosphatases confirmed that p52(S6K) was still regulated by phosphorylation. The sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein-serine phosphatase 2A and the heat shock protein 60. The association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A-specific antibodies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets.     

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.