Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha.
BACKGROUND
Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown.


RESULTS
We have purified 500 000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKBalpha at Thr308 and increases its activity over 30-fold. We tested the kinase in the presence of several inositol phospholipids and found that only low micromolar concentrations of the D enantiomers of either phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) or PtdIns(3,4)P2 were effective in potently activating the kinase, which has been named PtdIns(3,4,5)P3-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKBalpha or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. CONLCUSIONS: PDK1 is likely to be one of the protein kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4, 5)P3 and/or PtdIns(3,4)P2.
Alessi
D R
DR
Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN, Scotland. dralessi@bad.dundee.ac.uk
James
S R
SR
Downes
C P
CP
Holmes
A B
AB
Gaffney
P R
PR
Reese
C B
CB
Cohen
P
P
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity; D013912:Threonine; D014162:Transfection
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C494918:AKT1 protein, human; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt