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...

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