Activation of protein kinase B β and γ isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B α

Abstract

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBalpha, PKBbeta and PKBgamma) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, as well as PDK1. The activation of PKBbeta and PKBgamma by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBalpha, namely Thr309 (PKBbeta) and Thr305 (PKBgamma). PKBgamma which had been activated by PDK1 possessed a substrate specificity identical with that of PKBalpha and PKBbeta towards a range of peptides. The activation of PKBgamma and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBalpha and PKBbeta with similar kinetics. After stimulation of adipocytes, the activity of PKBbeta was twice that of PKBalpha, but in hepatocytes PKBalpha activity was four-fold higher than PKBbeta. Insulin induced the activation of PKBalpha in rat skeletal muscle in vivo, with little activation of PKBbeta. Insulin did not induce PKBgamma activity in adipocytes, hepatocytes or skeletal muscle, but PKBgamma was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects

“…In contrast, another PKB/Akt dominant-negative mutant, where the two phosphorylation sites had been mutated, did not prevent insulin-stimulated GLUT-4 translocation although other inhibitory effects were found [20]. The reason for these inconsistent results is not clear but could relate to the cells used, the complex regulation of PKB/Akt activity and cell-specific expression of the PKB/Akt isoforms [11,21]. A role for PKB/Akt is also supported by our recent findings with an inhibitor of PKB/Akt kinase activity which, albeit not selective, did not inhibit PI3-kinase activation.…”

“…The reason for this discrepancy is not clear. Human fat cells, similar to rat and differentiated 3T3-L1 adipocytes, mainly express the PKBb/Akt 2 isoform and hardly any PKBa/Akt 1 [23] whereas muscle predominantly expresses PKBa/Akt 1 and PKBg/Akt 3 [21]. Similar to PI3-kinase activation, possibly, however, of most importance is not total PKB/Akt activity in whole cells but the need for activation in the relevant subcellular compartments.…”

Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation

“…The phosphorylation sites are conserved in PKBβ (Thr$! *, Ser%(%), although, due to a C-terminal truncation, PKBγ lacks a Ser%($ homologue (PKBγ Thr$!& is in an analogous context to the P-loop site in PKBα) [50] (see Figure 1). …”

Nelfinavir-induced insulin resistance is associated with impaired plasma membrane recruitment of the PI 3-kinase effectors Akt/PKB and PKC-?

“…Anti p85, anti IRS-1, anti-phosphotyrosine (4G10), anti-protein phosphatase 2A (PP2A) antibodies and Ser/Thr Phosphatase Assay Kit 1 were supplied by Upstate Biotechnology (Lake Placid, N.Y., USA). AntiAkt2/PKB-β was a kind gift from Dr D. Alessi (University of Dundee, Scotland, United Kingdom) and was used as described previously [18]. Anti Na/K ATPase was a kind gift from Dr I. Sakler (Ben-Gurion University, Beer-Sheva, Israel).…”