PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

The substrate specificity of protein kinase-Bcc (PKB~ 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/ThrHyd, 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-ArgThr-Ser-Ser-Phe, was phosphorylated with a Km of 5 ttM and ma~ of 260 U/rag. PKBcx phosphorylated histone H2B (Km 5 ~NI, Vmax 68 U/rag) specifically at Ser-36 which also lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif. The peptide Arg-ProArg-Ala-Ala-Thr-Phe may be a relatively specific substrate for PKBcx because, unlike other substrates, it is not phosphorylated by p70 $6 kinase or MAP kinase activated protein (MAPKAP) kinase-1. . All forms of PKB possess an Nt;rminal pleckstrin homology (PH) domain, followed by a ttalytic domain and a short C-terminal tail. The catalytic c omain is most similar to cyclic AMP-dependent protein kixase (PKA, 65% similarity) and to protein kinase C (PKC, "5% similarity), findings that gave rise to two of its names, x amely PKB (i.e. between PKA and PKC) and RAC (Related 1 ) A and C kinase).PKBc~ is activated within 1 min following stimulation of tells with insulin [8,9]. The activation of PKBc~ by insulin , r IGF-1 is accompanied by its phosphorylation at Thr-308 and Ser-473, and phosphorylation of both of these residues is required to achieve a high level of activity [10]. Like the activation of PKB~ [11,12], the phosphorylation of Thr-308 and Ser-473 is prevented by wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase [10].The only physiological substrate for PKBc~ to have been identified to date is the protein kinase glycogen synthase kinase-3 (GSK3). GSK3 is inhibited in response to insulin with a half-time of 2 min, slightly slower than that for activation of PKBcx (1 min) and inhibition results from its phosphorylation at the same serine residue (Ser-21 in GSK3~ and Ser-9 in GSK3fi) which is targetted by PKB~x in vitro. Like the activation of PKBc~, the inhibition of GSK3 by insulin is prevented by the PI 3-kinase inhibitors wortmannin and LY 294002.GSK3cz and GSK3[3 are phosphorylated at Ser-21 and Ser-9, respectively, by two other insulin-stimulated protein kinases, namely p70 $6 kinase and MAP kinase-activated protein kinase-I (MAPKAP-KI, also known as p90 $6 kinase). However, these enzymes are not rate-limiting for the inhibition of GSK3 by insulin in L6 myotubes because specific inhibitors of their activation (rapamycin-p70 $6 kinase; PD 98059-MAPKAP kinase-1) have no effect [9]. The inhibition of GSK3 is thought to contribute to the stimulation of glycogen synthesis [9] and protein synthesis [13] by insulin.The activation of PI 3-kinase is essential for many of the effects of insulin and growth factors, including the stimulation of glucose transport, fatty...

No abstract

The ability of insulin to promote the phosphorylation of some proteins and the dephosphorylation of others is paradoxical. An insulin-stimulated protein kinase is shown to activate the type-1 protein phosphatase that controls glycogen metabolism, by phosphorylating its regulatory subunit at a specific serine. Furthermore, the phosphorylation of this residue is stimulated by insulin in vivo. Increased and decreased phosphorylation of proteins by insulin can therefore be explained through the same basic underlying mechanism.

The specificity of the catalytic subunit of protein phosphatase-1 (PP1,) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1,-binding domains on G, and G,, the subunits that target PPI, to hepatic and muscle glycogen, respectively, and on MI,", the subunit that targets PPI, to smooth muscle myosin. G,-(G63 -T93) interacted with PP1, and prevented G, from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate G, from PPI, or affect other characteristic properties of the PPlG, complex. These results indicate that G, contains two PP1,-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, G,-(G63 -N7.5) 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 G, from PP1 because phosphate is inserted into the PP1,-binding domain of G,. M,,<)-(Ml -E309) and Ml,,)-(MI -F38), but not Ml,,,-(D39-E309), mimicked the M,,, subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M,,,, subunit and Ml,~l-(MI -E309), neither Ml,,,-(MI -F38) nor M1,,-(D39-E309) suppressed the PPI,-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 M,,, 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. M,,,-(MI -F38) displaced G, from PPI,, while GM-(G63-T93) displaced M,,,, from PPlC in vitro. These observations indicate that the region(s) of PP1, that interact with G,/G, and MI,, overlap, explaining why different forms of PPI, contain just a single targetting subunit.Keywords: protein phosphatase-1 ; myosin ; smooth muscle ; glycogen metabolism.Protein phosphatase-1 (PPl), one of the major protein serine/ threonine phosphatases of eukaryotic cells, participates in the control of a variety of cellular functions that include glycogen metabolism, muscle contraction, the exit from mitosis (reviewed in [I, 21) and the splicing of mRNA 131. However, evidence has been accumulating that different processes are regulated by ~ Correspondence to P. Cohen, MRC Protein Phosphorylation Unit,

The peptide KKRNRTLTV, which combined these features, was relatively selective for p70s6K having a 50-fold higher V,,,,,/K,,, than MAPKAP kinase-1. Inactivation of the N-terminal kinase domain of MAPKAP kinase-1, which is 60% identical to p70S6K, abolished activity towards all peptides tested, but the enzyme retained 3040% of its activity if the C-terminal kinase domain kvas 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 MN/L/I-(RIK/H,X,X)-X-m-X-X-X-M/V/L/I.

Rabbits were starved for 24 h and injected with propranolol ( 2 mg/kg) with or without insulin (33 pg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol-treated animals the activity ratio (F glucose 6-phosphate) of glycogen synthase was 0.18 0.02 and the K, for glucose 6-phosphate was 1.2 0.1 mM. In (propranolol + insuIin)-treated animals the activity ratio was 0.35 & 0.02 and the I(, for glucose 6-phosphate was 0.60 & 0.05 mM.0.09 niol 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 (la, lb, 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 v i m 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-AMPdependent protein kinase (la, I b 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.Glycogen synthase purified to homogeneity from propranolol-treated animals contained 2.74Glycogen synthase is the best documented and one of the most complex examples of 'multisite phosphorylation', a phenomenon that is being encountered with increasing frequency [I]. The enzyme is phosphorylated on seven serine residues by at least five different protein kinases [2] [7].In general, phosphorylation increases the K , for UDPglucose, decreases the Ki for inhibitors such as Pi and ADP, and increases the K, for the activator glucose-6-P [S]. Phosphorylation of sites (3a + 3b + 3c) or site 2 influences the kinetic parameters to a greater extent than site l a , but the ~ Abbreviations. SDS, sodium dodecyl sulphate; HEDTA, N '-(2-hydroxyethyl)-ethylenedianiine-N,N',N-triacetate; HPLC, high-performance liquid chromatography ; glucose-6-P, glucose 6-phosphate.Enzymes. Phosphorylase (EC 2.4.1.1); phosphorylase kina...