Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2.

The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except that it could tolerate replacement of the arginine by a lysine and the phosphorylation-site serine by a threonine residue. Partial cDNAs encoding MAPKAP kinase-2 were isolated from rabbit and human skeletal muscle and human teratocarcinoma libraries, and Northern-blotting experiments revealed a single 3.3 kb mRNA transcript present at similar levels in six human tissues examined. The catalytic domain was most similar (35-40% identity) to calmodulin-dependent protein kinases II and IV, phosphorylase kinase, putative serine kinase H1 and the C-terminal domain of MAPKAP kinase-1, which form one branch of the protein kinase phylogenetic tree. The sequence N-terminal to the catalytic domain is proline-rich and contains two putative SH3-binding sites. The threonine residue phosphorylated by MAP kinase lies immediately C-terminal to the catalytic domain and is followed by a nuclear localization signal, Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys, near the C-terminus.

Differential phosphorylation of ribosomal protein S6 in isolated rat hepatocytes after incubation with insulin and glucagon.

Glucagon and insulin both stimulated the 32P-labelling of ribosomal protein S6 in rat hepatocytes that had been incubated with 32Pi. Glucagon selectively enhanced the labelling of the tryptic peptide phosphorylated by cyclic AMP-dependent protein kinase, demonstrating that 6 S is a physiological substrate for this enzyme. Insulin stimulated the phosphorylation of distinct tryptic peptides, at least one of which appears to be very close in the primary structure to the sites phosphorylated by cyclic AMP-dependent protein kinase.