Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes.
By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt.
Hresko
Richard C
RC
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
Murata
Haruhiko
H
Mueckler
Mike
M
D064413:3-Phosphoinositide-Dependent Protein Kinases; D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D003599:Cytoskeleton / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000199:Actins; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D013912:Threonine; C578562:PDPK1 protein, human; C578564:Pdpk1 protein, mouse