Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKC, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKC interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKC, leading to phosphorylation of the PKB-PH domain on Thr 34 . This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP 3 even in the presence of a ceramide-activated PKC and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKC and a kinase-dead PKC both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKC-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.Protein kinase B (PKB), also known as c-Akt, is a serine/ threonine kinase that has been implicated in the control of diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis (16,27,34,48). Three PKB isoforms (␣, ␤, and ␥) have been identified, and these can be activated rapidly in response to insulin and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. PI3K activation results in the increased production of 3-phosphoinositides, e.g., phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4-bisphosphate, which play a key role in the recruitment of PKB to the plasma membrane (5). The N-terminal domain of all three PKB isoforms contains a pleckstrin homology (PH) domain, which is considered critical in allowing the kinase to interact with 3-phosphoinositides and possibly other signaling proteins (13,16,19). Binding of 3-phosphoinositides to the PH domain of PKB is also thought to induce conformational changes in the kinase that expose two key regulatory sites, Thr 308 and Ser 473(3), allowing them to be phosphorylated by two upstream kinases. One of these, 3-phosphoinositide-dependent kinase-1 (PDK1), phosphorylates Thr 308 (4, 44), whereas the identity of the second kinase that phosphorylates Ser 473 (putatively termed PDK2) remains unknown, although a number of potential candidates have recently been proposed (for a review, see reference 15).The activation of PKB elicited by insulin and g...

The E3 ubiquitin ligase Mdm2 is a focal regulator of p53 tumour suppressor activity. It binds p53, promoting its polyubiquitination and degradation, and also controls p53 synthesis. However, it is not known how this dual function of Mdm2 on p53 synthesis and degradation is achieved. Here we show that the p53 mRNA region encoding the Mdm2-binding site interacts directly with the RING domain of Mdm2. This impairs the E3 ligase activity of Mdm2 and promotes p53 mRNA translation. We also show that introduction of cancer-derived single silent point-mutations in the p53 mRNA weakens its binding to Mdm2 and results in reduced p53 activity. These data are consistent with a mechanism by which changes in silent nucleotides can affect the function of the encoded protein, and indicate that Mdm2-mediated control of p53 synthesis and degradation has evolved in the p53 mRNA sequence and its encoded amino acids.

Non-esterified fatty acids (NEFAs) have been implicated in the pathogenesis of skeletal muscle insulin resistance that may develop, in part, as a consequence of a direct inhibitory effect on early insulin signalling events. Here we report work investigating the mechanism by which palmitate (a saturated free fatty acid) inhibits insulin action in rat L6 myotubes. Palmitate suppressed the insulin-induced plasma membrane recruitment and phosphorylation of protein kinase B (PKB) and this was associated with a loss in insulin-stimulated glucose transport. The inhibition in PKB was not due to a loss in insulin receptor substrate (IRS)1 tyrosine phosphorylation, IRS-1/p85 (phosphoinositide 3-kinase) association or suppression in phosphatidyl 3,4,5 triphosphate synthesis, but was attributable to an elevated intracellular synthesis of ceramide (6-fold) from palmitate and a concomitant activation of protein kinase PKCzeta (5-fold). Inhibitors of serine palmitoyl transferase suppressed the intracellular synthesis of ceramide from palmitate, prevented PKCzeta activation, and antagonized the inhibition in PKB recruitment/phosphorylation and the loss in insulin-stimulated glucose transport elicited by the NEFA. Inhibiting the palmitate-induced activation of PKCzeta with Ro 31.8220, also prevented the loss in the insulin-dependent phosphorylation of PKB caused by palmitate. These findings indicate that intracellular ceramide synthesis and PKCzeta activation are important aspects of the mechanism by which palmitate desensitizes L6 muscle cells to insulin.