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Mitogen-activated protein kinase-activated protein kinase-1 (MAPKAP-K1; also known as p90 rsk ) contains two protein kinase domains in a single polypeptide. The N-terminal kinase domain is necessary for the phosphorylation of peptide substrates, whereas the C-terminal kinase domain is required for full activation of the N-terminal domain. Here we identify six sites in MAPKAP-K1a that become phosphorylated in transfected COS-1 cells. The inactive form of MAPKAP-K1a in unstimulated cells is partially phosphorylated at Ser222 and Ser733. Stimulation with phorbol 12-myristate 13-acetate induces the phosphorylation of Thr360, Ser364, Thr574, and Ser381 and increases the phosphorylation of Ser222 and Ser733. Our data indicate that mitogen-activated protein kinase activates the C-terminal kinase domain by phosphorylating Thr574 and contributes to the activation of the N-terminal kinase domain by phosphorylating Ser364. The activated C-terminal domain phosphorylates Ser381, which, together with phosphorylation of Ser364, activates the N-terminal kinase domain. The phosphorylation of Ser222and Ser733, which can be catalyzed by the N-terminal domain, does not activate MAPKAP-K1a per se, but Ser222 phosphorylation appears to be required for activation. Ser222, Ser364, and Ser381 are situated in analogous positions to phosphorylation sites in protein kinase B, protein kinase C, and p70S6K, suggesting a common mechanism of activation for these growth factor-stimulated protein kinases.

Background: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ). Results: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p 13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB α only in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . Overexpression of PDK1 in 293 cells activated PKB α and potentiated the IGF1-induced phosphorylation of PKB α at Thr308. Experiments in which the PH domains of either PDK1 or PKB α were deleted indicated that the binding of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 to PKB α is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. Conclusions: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 in the activation of PKB α is at least partly substrate directed.

The substrate specificity of protein kinase-B alpha (PKBalpha, also known as RAC kinase or Akt) was investigated using synthetic peptide substrates related to the sequence surrounding the phosphorylation site on glycogen synthase kinase-3 (GSK3). The minimum sequence motif required for efficient phosphorylation was Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hyd, where Xaa is any amino acid, Yaa and Zaa are small residues other than glycine and Hyd is a bulky hydrophobic residue (Phe, Leu). The most effective substrate, Arg-Pro-Arg-Thr-Ser-Ser-Phe, was phosphorylated with a Km of 5 microM and Vmax of 260 U/mg. PKBalpha phosphorylated histone H2B (Km 5 microM, Vmax 68 U/mg) specifically at Ser-36 which also lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif. The peptide Arg-Pro-Arg-Ala-Ala-Thr-Phe may be a relatively specific substrate for PKBalpha because, unlike other substrates, it is not phosphorylated by p70 S6 kinase or MAP kinase activated protein (MAPKAP) kinase-1.

The in vivo phosphorylation stoichiometries of 4 serines on the glycogen-binding (G)-subunit of protein phosphatase 1 (PP1) have been determined. In fed rabbits injected with propranolol stoichiometries (mol/mol) were: site 1 (0.67 +/- 0.09), site 2 (0.20 +/- 0.07), site 3a (0.23 +/- 0.01) and site 3b (0). After injection with adrenalin they became: site 1 (0.90 +/- 0.02), site 2 (0.72 +/- 0.01), site 3a (0.23 +/- 0.02) and site 3b (0). These results, together with other data, establish that site 2 phosphorylation by cyclic AMP-dependent protein kinase triggers dissociation of PP1 from the G-subunit in vivo. They also demonstrate that a residue phosphorylated in vitro by glycogen synthase kinase 3 (site 3a) is phosphorylated in vivo.

The specificity of the catalytic subunit of protein phosphatase-1 (PP1c) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1c-binding domains on GL and GM, the subunits that target PP1c to hepatic and muscle glycogen, respectively, and on M110, the subunit that targets PP1c to smooth muscle myosin. GM-(G63-T93) interacted with PP1c and prevented GL from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate GL from PP1c or affect other characteristic properties of the PP1GL complex. These results indicate that GL contains two PP1c-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, GM-(G63-N75) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates GM from PP1c because phosphate is inserted into the PP1c-binding domain of GM. M110-(M1-E309) and M110-(M1-F38), but not M110-(D39-E309), mimicked the M110 subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M110 subunit and M110-(M1-E309), neither M110-(M1-F38) nor M110-(D39-E309) suppressed the PP1c-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M110 subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M110-(M1-F38) displaced GL from PP1c, while GM-(G63-T93) displaced M110 from PP1c in vitro. These observations indicate that the region(s) of PP1c that interact with GM/GL and M110 overlap, explaining why different forms of PP1c contain just a single targetting subunit.

xxR/KxRxxSxx sequences were phosphorylated with high efficiency by both p70 S6 kinase (p70 S6K ) and MAPKAP kinase-1. The best substrate for MAPKAP kinase-1 (KKKNRTLSVA) was phosphorylated with a K m of 0.17 μM, and the best substrate for p70 S6K (KKRNRTLSVA) with a K m of 1.5 μM. The requirement of both enzymes for Arg/Lys at position n -5 could be partially replaced by inserting basic residues at other positions, especially by an Arg at n - 2 or n - 4. MAPKAP kinase-1 (but not p70 S6K ) tolerated lack of any residue at n - 5 if Arg was present at n - 2 and n - 3. p70 S6K (but not p90 S6K ) tolerated Thr at position n and absence of any residue at n + 2. The peptide KKRNRTLTV, which combined these features, was relatively selective for p70 S6K having a 50-fold higher V max / K m than MAPKAP kinase-1. Inactivation of the N-terminal kinase domain of MAPKAP kinase-1, which is 60% identical to p70 S6K , abolished activity towards all peptides tested, but the enzyme retained 30–40% of its activity if the C-terminal kinase domain was inactivated.

Inspection of sequences around sites phosphorylated by the AMP-activated protein kinase (AMP-PK), and homologous sequences from other species, indicates conserved features. There are hydrophobic residues (M, V, L, I) at P-5 and P+4, and at least one basic residue (R, K, H) at P-2, P-3 or P-4. The importance of these residues has been established for AMP-PK and its putative plant homologue using a series of synthetic peptides. These results confirm the functional similarity of the animal and plant kinases, and suggest that the required motif for recognition of substrate by either kinase is M/V/L/I-(R/K/H,X,X)-X-S/T-X-X-X-M/V/L/I.

The amino acid sequence of a region on the glycogen-binding (G)-subunit of protein phosphatase-1G that is phosphorylated by cyclic AMP-dependent protein kinase has been determined. The sequence is: (formula see text) This finding will facilitate studies of the effects of hormones on the phosphorylation state of the G-subunit in vivo.

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in λgt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1–101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.

Rabbits were starved for 24 h and injected with propranolol (2 mg/kg) with or without insulin (33 μg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol-treated animals the activity ratio (± glucose 6-phosphate) of glycogen synthase was 0.18 ± 0.02 and the Ka for glucose 6-phosphate was 1.2 ± 0.1 mM. In (propranolol + insulin)-treated animals the activity ratio was 0.35 ± 0.02 and the Ka for glucose 6-phosphate was 0.60 ± 0.05 mM. Glycogen synthase purified to homogeneity from propranolol-treated animals contained 2.74 ± 0.09 mol phosphate/mol subunit, whereas this value was 2.33 ± 0.09 mol phosphate/mol subunit from (propranolol + insulin)-treated animals. The distribution of phosphate between the seven phosphorylation sites was elucidated. In (propranolol + insulin)-treated animals there was a decrease of 0.4—0.45 mol phosphate/mol subunit in sites (3a + 3b + 3c), and no significant changes in any other site (1a, 1b, 2 and 5). The results demonstrate that dephosphorylation of sites (3a + 3b + 3c) is responsible for the activation of glycogen synthase observed after acute administration of insulin. Neither protein phosphatase 1 nor protein phosphatase 2A, which account for virtually all the glycogen synthase phosphatase activity in skeletal muscle extracts, were able to mimic the specific dephosphorylation of sites (3a + 3b + 3c) produced by insulin. Both enzymes dephosphorylated site 2 and sites (3a + 3b + 3c) in vitro at comparable rates. The action of insulin may therefore involve a decrease in the activity of glycogen synthase kinase 3 and the regulation of this enzyme is discussed. The failure of insulin to produce a significant dephosphorylation of the sites phosphorylated by cyclic-AMP-dependent protein kinase (1a, 1b and 2) demonstrates that inhibition of this protein kinase does not underlie the activation of glycogen synthase by this hormone. This conclusion is supported by measurements of the phosphorylation states of inhibitor 1, phosphorylase kinase and phosphorylase. Neither 24-h starvation, nor alloxon-induced diabetes, produced significant changes in the kinetic properties or phosphorylation state of glycogen synthase. Possible reasons for these findings are discussed.

1,4-alpha-glucan:1,4-alpha-glucan 6-alpha-D-(1,4-alpha-D-glucano) transferase (branching enzyme) was purified by ammonium sulphate precipitation, chromatography on DEAE-cellulose, fractionation with poly(ethyleneglycol) 6000, chromatography on DEAE-Sepharose and gel filtration on Sephadex G150. The final specific activity was 3000 U/mg corresponding to a purification of approximately 10000-fold over the muscle extracts. 0.6 mg of enzyme was isolated from 4000 g muscle within eight days corresponding to an overall yield of 7%. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, and this technique yielded a molecular weight of 77000 for the subunit molecular weight of branching enzyme. The apparent molecular weight of the native enzyme determined by gel filtration on Sephadex G150 was 60000, demonstrating that branching enzyme is a monomeric protein. Only a very small proportion of the branching enzyme activity in muscle extracts (2%) precipitated with the protein-glycogen complex. This finding, and its low concentration in muscle, explain why a protein-staining band corresponding to branching enzyme cannot be detected by polyacrylamide gel electrophoresis of the protein-glycogen complex.

The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.

Incubation of liver extracts with MgATP led to complete inactivation of the aminoacyl-tRNA synthetase complex provided that sodium fluoride was included. If sodium fluoride was omitted no inactivation was observed. The arginyl-tRNA and isoleucyl-tRNA synthetases were inactivated more slowly than the leucyl-tRNA, lysyl-tRNA and methionyl-tRNA synthetases. Gel filtration on Sepharose 4B separated the aminoacyl-tRNA synthetase complex (Ve/Vo= 1.2) from an inactivating factor that required MgATP (Ve/Vo= 2.8). The inactivated aminoacyl-tRNA synthetase complex was eluted from Sepharose-4B at the same position as the activated complex, and was resolved from a reactivating factor that required Mg2+ and was inhibited by sodium fluoride (Ve/Vo= 2.5). The arginyi-tRNA and isoleucyl-tRNA synthetases were reactivated more rapidly than the leucyl-tRNA, lysyl-tRNA and methionyl-tRNA synthetases. The aminoacyl-tRNA synthetase complex could also be reactivated using highly purified preparations of either protein phosphatase 1 or protein phosphatase 2A. Inhibitor 2, a specific inhibitor of protein phosphatase 1, prevented reactivation of the complex by protein phosphatase 1, but not by protein phosphatase 2A. The relative rates of reactivation of the different activities of the complex by either protein phosphatase 1 or protein phosphatase 2A were very similar to those observed with the reactivating factor. The aminoacyl-tRNA synthetase complex was largely in the activated state in livers of normally fed rats, but overnight starvation produced substantial conversion to the inactivated form of the complex. Incubation of hepatocytes from normally fed rats with glucagon and isobutylmethylxanthine for 20 min also caused a substantial decrease in the activation state from approximately 80% to approximately 30%. Conversely, when hepatocytes prepared from starved rats were incubated with insulin for 30 min, almost complete reactivation of the complex was achieved. The results indicate that the aminoacyl-tRNA synthetase complex is regulated by a phosphorylation dephosphorylation mechanism in vitro and in vivo. The effects of hormones on the activation state of the complex are discussed in the light of the observation that the inactivating factor is not cyclic-AMP-dependent protein kinase.