PDK-1 activation is a key event in the intracellular signaling triggered by a number of hormones, including insulin (3
). There is evidence that insulin induces tyrosine phosphorylation of PDK-1 (30
), which may contribute to its subcellular redistribution and activation. However, the molecular mechanism through which insulin triggers PDK-1 activation, as well as the kinase responsible for PDK-1 phosphorylation in the insulin-stimulated cells, remains elusive. In the present work, we report that insulin induces phosphorylation of PDK-1 on tyrosines 9 and 373/376 and activation of PDK-1 in cultured L6 skeletal-muscle cells, as well as in mouse skeletal muscle and adipose tissue. Consistent with previous studies by Park et al. (30
), these tyrosine phosphorylation events are necessary for insulin metabolic action, as expression of Tyr→Phe-substituted PDK-1 blocks insulin stimulation of both glucose uptake and glycogen synthase in L6 cells, like treatment with a specific PDK-1 siRNA. In the cell extracts from these cells, insulin also induces the coprecipitation of insulin receptors with PDK-1. The coprecipitation did not occur in cell extracts expressing the hIRΔ43
C-terminally truncated receptor, indicating that PDK-1 binds to the active insulin receptor kinase and that the 43 C-terminal amino acids of the insulin receptor are necessary for the binding to occur.
Consistent with earlier findings in different (26
), though not all, cell types (29
), we have also shown that the hIRΔ43
receptor is unable to transduce the insulin stimulatory effect on glucose transport and glycogen synthase in L6 cells while conveying proliferative stimuli. In parallel, there was little insulin-dependent tyrosine phosphorylation and activation of PDK-1 in L6 cells expressing hIRΔ43
receptors. Thus, binding to the active insulin receptor kinase is accompanied by PDK-1 tyrosine phosphorylation and activation.
Src family tyrosine kinases have been implicated in insulin signaling and shown to phosphorylate PDK-1 on tyrosine in certain cell types (20
). However, Src kinases do not mediate insulin action on PDK-1 tyrosine phosphorylation in L6 cells, as Src kinase inhibitors have no effect on PDK-1 phosphorylation by insulin. The insulin receptor kinase itself appears to phosphorylate on tyrosine and to activate PDK-1 in skeletal-muscle cells. In fact, (i) as in the intact cells, the wild-type but not the hIRΔ43
receptors bind and phosphorylate recombinant PDK-1 in vitro, and (ii) a peptide corresponding to the 43-amino-acid sequence of the insulin receptor C terminus (Δ43 peptide) simultaneously prevents insulin receptor interaction and phosphorylation of PDK-1 and PDK-1 activation, both in vitro and in intact cells. The additional finding that the Δ43 peptide coprecipitates with PDK-1 only in extracts from insulin-stimulated cells indicates that PDK-1 interaction requires phosphorylation of the insulin receptor C-terminal tyrosines. Indeed, we showed that (i) Tyr→Phe substitution in the Δ43 peptide abolished its ability to inhibit both the insulin-induced IR-PDK-1 interaction and PDK-1 tyrosine phosphorylation and (ii) the Δ43 peptide undergoes tyrosine phosphorylation by insulin (data not shown).
Previous reports demonstrated that the binding of PI 3-K lipids to the PH domain of PDK-1 targets PDK-1 to the plasma membrane, enabling PDK-1 activation and glucose uptake (2
). How PDK-1 membrane interaction affects kinase activity is poorly defined, however. As previously demonstrated in other cells, we reported that blocking of PI 3-K in insulin-exposed L6 cells prevents PDK-1 membrane translocation and activation. In addition to inducing PDK-1 activity, however, we further showed that activated insulin receptors anchor the membrane-targeted PDK-1 at the cell surface. Indeed, (i) membrane-targeted PDK-1 in L6 cells expressing hIRΔ43
receptors retrotranslocate to the cytosol significantly faster than in cells expressing wild-type receptors, and (ii) blocking of wild-type insulin receptor binding with the Δ43 peptide induces fast cytosolic translocation of PDK-1 in insulin-stimulated L6 cells, accompanied by reduced insulin-stimulated metabolic effects. When cells are exposed to multiple hormones in the extracellular fluid, the persistence of PDK-1 in the membrane due to activated insulin receptors may prolong PDK-1 activity. Indeed, PDK-1 activation in response to platelet-derived growth factor shows a shorter time course in L6 cells expressing hIRΔ43
than in cells expressing wild-type receptors (data not shown). Thus, PDK-1 membrane anchoring by the insulin receptor may also affect response to hormones other than insulin.
Mutants of PDK-1 unable to translocate to the plasma membrane prevent membrane recruitment and subsequent activation of Akt/PKB in insulin-stimulated cells (2
). Based on this finding, it has been proposed that PDK-1 recruits Akt/PKB to the plasma membrane, where the activation of Akt/PKB occurs (14
). However, in L6 cells expressing hIRΔ43
receptors, Akt/PKB normally translocates following insulin exposure, despite the lack of PDK-1 activation and the reduced presence of PDK-1 in the membrane. It is possible that insulin activation of the endogenous receptors in the hIRΔ43
transfectants is sufficient to allow maximal translocation of Akt/PKB. Alternatively, PI 3-K and/or PDK-2 activation may be sufficient for Akt/PKB recruitment in the hIRΔ43
-transfected cells. Indeed, previous studies by Williams et al. (45
) demonstrated that, in cells lacking PDK-1, growth factor-stimulated phosphorylation of Akt/PKB on Thr308
did not occur, but phosphorylation of Ser473
still remained intact. Consistently, insulin-dependent phosphorylation of Ser473
was unaffected in the hIRΔ43
In conclusion, we report, for the first time that the insulin receptor kinase binds to and tyrosine phosphorylates PDK-1 in response to insulin (thereby activating PDK-1). Membrane anchoring of PDK-1 through the C-terminal 1340-to-1382 region of the insulin receptor is a crucial step in insulin metabolic action in skeletal-muscle cells and may affect PDK-1 signaling in response to other hormones as well.