Ser-474 is the major target of insulin-mediated phosphorylation of protein kinase B b in primary rat adipocytes Olga Go ¨ransson a, *, Svante Resjo ¨ a , Lars Ro ¨nnstrand b , Vincent Manganiello c , Eva Degerman a a Section for Molecular Signalling, Department of Cell and Molecular Biology, Lund University, BMC, C11, S-221 84 Lund, Sweden b Ludwig Institute for Cancer Research, Biomedical Center, PO Box 595, S-751 24 Uppsala, Sweden c Pulmonary/Critical Care Medicine Branch, National Heart, Lung and Blood Institutes, Bethesda, MD 20892, USA Received 5 June 2001; accepted 14 August 2001 Abstract The mechanism of activation for protein kinase B (PKB), an important target for insulin signaling, has been scarcely investigated in primary cells. In this study, we have characterized the insulin-induced phosphorylation and activation of PKBb in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKBb from cytosol to membranes, and phosphorylation and activation of PKBb. Phosphoamino acid analysis and phosphopeptide mapping demonstrated that the phosphorylation occurred mainly on serines, also when using calyculin A, and that these were localized within one major phosphopeptide. Radiosequencing showed that the radioactivity was released in Cycle No. 7. In addition, the peptide was specifically immunoprecipitated from a tryptic digest of PKBb using the anti-phospho- PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKBb mainly is phosphorylated on Ser-474 in response to insulin stimulation, in contrast to previous studies in human embryonic kidney (HEK) 293 cells demonstrating, in addition, phosphorylation of Thr-309. D 2002 Elsevier Science Inc. All rights reserved. Keywords: PKBb; Adipocyte; Phosphorylation; Insulin; Translocation; Phosphopeptide 1. Introduction In recent years, the serine/threonine (Ser/Thr) kinase protein kinase B (PKB), or Akt, has been identified as an important downstream target for signals that activate phos- phoinositide 3-kinase (PI3-K), such as insulin and various growth factors [1,2]. The role of PKB in different biological responses to these stimuli has been extensively studied, and PKB has so far been implicated in events such as glucose uptake, glycogen synthesis, protein synthesis and cell sur- vival [1,2]. PKB is expressed as three isoforms, PKBa [3–5], -b [6] and -g [7], of which a and b are widely distributed, whereas the g form is expressed primarily in brain and testis. Studies, mainly performed in cultured cell lines overex- pressing PKB, aimed at elucidating the mode of activation of PKB, have led to a complex and not yet complete picture of how PKB is regulated in response to activators of PI3-K. The lipid products of activated PI3-K, mainly phosphatidy- linositol(3, 4, 5)-trisphosphate (PIP 3 ), bind to the N-termin- ally located pleckstrin homology (PH) domain of PKB, thereby causing the otherwise cytosolic kinase to translocate to the plasma membrane [8–10]. This is believed to bring PKB in close proximity to upstream kinases, which then phosphorylate and activate the enzyme. The binding of PKB to the membrane may also induce a conformational change in the protein, rendering it more susceptible to phosphor- ylation. In L6 myotubes, and in human embryonic kidney (HEK) 293 cells, the cell type that to the largest extent has 0898-6568/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII:S0898-6568(01)00242-X Abbreviations: HEK, human embryonic kidney; 2D-PAAA, two- dimensional phosphoamino acid analysis; PAGE, polyacrylamide gel electrophoresis; PDE 3B, phosphodiesterase 3B; PDK1, PIP 3 -dependent protein kinase-1; PH, pleckstrin homology; PIF, PDK1-interacting frag- ment; PKB, protein kinase B; PI3-K, phosphoinositide 3-kinase; PIP 3 , phosphatidylinositol(3, 4, 5)-trisphosphate; pV, peroxovanadate; P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine * Corresponding author. Tel.: +46-46-222-8984; fax: +46-46-222- 4022. E-mail address: olga.goransson@medkem.lu.se (O. Go ¨ransson). www.elsevier.com/locate/cellsig Cellular Signalling 14 (2002) 175 – 182