Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts.

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

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.     

Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.     

Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells.

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.     

Phosphorylation of the insulin receptor by a casein kinase I-like enzyme.

A serine protein kinase that phosphorylates the beta-subunit of the insulin receptor has been partially purified 5,000-fold from HeLa cell membranes. The enzyme has been purified by ion-exchange and hydroxylapatite chromatography and sucrose gradient centrifugation; it has an apparent molecular weight of 36,000-43,000 daltons. It exhibits the following properties: (a) it catalyzes the phosphorylation of the autophosphorylated insulin receptor more efficiently than the nonautophosphorylated insulin receptor, (b) it decreases insulin receptor phosphorylation of tubulin but has no effect on insulin receptor phosphorylation of microtubule-associated proteins or reduced and carboxyamidomethylated lysozyme. The enzyme also phosphorylates casein and ribosomal protein S6 and shares many properties with casein kinase I: (a) similar molecular weight, (b) utilization of ATP but not GTP as phosphoryl donor, and (c) sensitivity to inhibition by heparin. Based on several criteria the receptor serine kinase is neither protein kinase C nor the cAMP-dependent protein kinase.     

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.     

Characterization of a mitogen-activated, Ca2+-sensitive microtubule-associated protein-2 kinase

We have previously found that treatment of quiescent mammalian fibroblast cells with several mitogenic factors activates in common a Ca2+-sensitive serine/threonine-specific protein kinase activity toward microtubule-associated protein 2 (MAP2) [Hoshi, M., Nishida, E. and Sakai, H. (1988) J. Biol. Chem. 263, 5396-5401]. Here, we characterized the mitogen-activated MAP2 kinase activity in rat 3Y1 cells. The activated kinase activity was detected in the cytosolic fraction but not in the membrane fraction. The inhibitory effect of Ca2+ on the kinase activity was reversible. Kinetic analyses revealed that the apparent Km values of the kinase activity for MAP2 and ATP were 1.6 microM and 30 microM, respectively. Free Ca2+ at 4 microM decreased apparent Vmax values for MAP2 and ATP without changing the apparent Km values. The MAP2 kinase had an apparent molecular mass of about 40 kDa as determined by gel filtration and by sucrose density gradient centrifugation. Myelin basic protein as well as MAP2 could serve as good substrates for this kinase, but 40S ribosomal protein S6, casein, histone, phosphorylase b, protamine, tubulin, actin and tau could not. These properties of the enzyme indicate that the Ca2+-sensitive MAP2 kinase may be a previously unidentified enzyme. Down-regulation of protein kinase C by prolonged phorbol ester treatment abolished the MAP2 kinase activation by phorbol ester, but did not prevent the MAP2 kinase activation by epidermal growth factor (EGF) or fresh serum. This suggests that the Ca2+-sensitive MAP2 kinase could be activated through protein-kinase-C-dependent and -independent pathways. Activation of the MAP2 kinase occurred shortly after the addition of EGF or phorbol ester even in the presence of protein synthesis inhibitors (cycloheximide, puromycin and emetin). Moreover, treatment of the EGF- or phorbol-ester-activated MAP2 kinase with acid phosphatase inactivated the kinase activity. Thus, the MAP2 kinase may be activated through phosphorylation.     

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.     

Activation of microtubule-associated protein kinase by microtubule disruption in quiescent rat 3Y1 cells.

Treatment of quiescent rat fibroblastic cells (3Y1) with colchicine, a microtubule-disrupting agent, which could induce the initiation of DNA synthesis [Y. Shinohara, E. Nishida, and H. Sakai (1989) Eur. J. Biochem. 183, 275-280], activated a serine/threonine-specific protein kinase activity in cell extracts that preferentially phosphorylated exogenous microtubule-associated protein 2 (MAP2). Vinblastine treatment also activated the kinase activity, and taxol pretreatment inhibited the colchicine-induced activation of this kinase activity. The detailed biochemical characterization indicated that this microtubule disruption-activated MAP2 kinase was very similar or identical to the mitogen-activated MAP kinase in the substrate specificity and chromatographic behaviors on phosphocellulose, DEAE-cellulose, gel filtration, and phenyl-Sepharose. Pretreatment of the cells with protein synthesis inhibitors did not prevent the MAP2 kinase activation by colchicine. Moreover, phosphatase treatment inactivated the colchicine-activated MAP2 kinase activity. These data suggest that microtubule disruption activates MAP kinase through phosphorylation.     

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.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.     

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.     

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

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

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.     

Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate.

Treatment of BC3H1 myocytes or 3T3-L1 fibroblasts with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42-kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from growth factor-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within 15 min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by growth factors in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase.     

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

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

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

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

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

The sites at which brain microtubule-associated protein 2 is phosphorylated in vivo differ from those accessible to cAMP-dependent kinase in vitro

The most conspicuous brain microtubule-associated protein, MAP-2, has been shown to contain 8-10 mol of covalently bound phosphate/mol, as isolated. The MAP-2-associated cAMP-dependent protein kinase can add 10-12 more phosphates, using cycled microtubule preparations, but it does not catalyze exchange between ATP and the pre-existing protein phosphate. We now show that the phosphates that turn over in vivo, after intracerebral injection of 32Pi, are primarily in the projection domain of MAP-2, whereas the sites phosphorylated in vitro are more concentrated in the binding domain. Phosphoserine and phosphothreonine were recovered in a 6:1 ratio from partial acid hydrolysates of MAP-2 phosphorylated either in vivo or in vitro. A protein phosphatase, purified from brain, released residues from in vitro sites in both domains. The enzyme did not release appreciable phosphate that had turned over in vivo, and similar specificity was shown by three other purified protein phosphatases: calcineurin (also from brain) and smooth muscle phosphatases I and II. Thus, even in the projection domain, different sites may be involved.