P53 mRNA controls p53 activity by managing Mdm2 functions.

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.

Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism.

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 PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta 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 PKCzeta, 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 PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta 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 PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.

Ceramide down-regulates System A amino acid transport and protein synthesis in rat skeletal muscle cells.

Skeletal muscle is a major insulin target tissue and has a prominent role in the control of body amino acid economy, being the principal store of free and protein-bound amino acids and a dominant locus for amino acid metabolism. Interplay between diverse stimuli (e.g., hormonal/nutritional/mechanical) modulates muscle insulin action to serve physiological need through the action of factors such as intramuscular signaling molecules. Ceramide, a product of sphingolipid metabolism and cytokine signaling, has a potent contra-insulin action with respect to the transport and deposition of glucose in skeletal muscle, although ceramide effects on muscle amino acid turnover have not previously been documented. Here, membrane permeant C2-ceramide is shown to attenuate the basal and insulin-stimulated activity of the Na+-dependent System A amino acid transporter in rat muscle cells (L6 myotubes) by depletion of the plasma membrane abundance of SNAT2 (a System A isoform). Concomitant with transporter down-regulation, ceramide diminished both intramyocellular amino acid abundance and the phosphorylation of translation regulators lying downstream of mTOR. The physiological outcome of ceramide signaling in this instance is a marked reduction in cellular protein synthesis, a result that is likely to represent an important component of the processes leading to muscle wasting in catabolic conditions.

Intracellular ceramide synthesis and protein kinase Czeta activation play an essential role in palmitate-induced insulin resistance in rat L6 skeletal muscle cells.

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.

Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes.

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

Stress-dependent changes in the properties of p53 complexes by the alternative translation product p53/47.

P53 plays a key role in the cellular response to damage exposure and in preventing the development of human cancers. Activation of p53 results in changes in the expression of a large number of gene products. However, relatively little is still known how p53 activation differentiates between different types of damages in different types of tissues or how this triggers either an apoptotic response or cell cycle arrest and DNA repair. The p53 message is translated into two products with distinct activities and stabilities through alternative mechanisms of initiation. P53/47 is initiated 40 codons down stream of the full length p53 and does not include the binding site for the E3 ubiquitin ligase Mdm2 or the transactivation domain I but retains the capacity to form p53 hetero- and homo-oligomers. Here we report that p53/47 controls the folding, the oligomerisation and the post-translational modification of p53 complexes and that it diversifies p53 properties in a cell stress-dependent fashion. P21 expression, for example, is under normal conditions not affected by p53/47 but is induced 18-fold after treatment of cells with the DNA damaging drug doxorubicin. This is accompanied by the recruitment of p53/47 to the p21 promoter.

The novel platinum(IV) complex LA-12 induces p53 and p53/47 responses that differ from the related drug, cisplatin.

The platinum(II)-based complex cisplatin is one of the most frequently used antitumour agents; however, a high incidence of harmful side effects and the frequent emergence of acquired resistance are the major clinical problems. The novel platinum(IV)-based complex LA-12 exhibits a high efficacy against cancer cell lines, including cisplatin-insensitive cells, but the mechanisms by which LA-12 perturbs cell growth are unclear. We tested the effects of LA-12 on the p53 response and demonstrate that LA-12 induces unique changes in the profile of gene expression compared with cisplatin and doxorubicin. Furthermore, the ability of LA-12 to disrupt cellular proliferation is greatly enhanced by the expression of p53 and p53/47 indicating both p53-dependent and p53-independent effects of LA-12. Exposure of the human cancer cell lines H1299, A2780, BT549 and BT474 to LA-12 alters the expression of p53 and p53/47 in both a time-dependent and dose-dependent manner. Treatment of cells with a low concentration of the drug results in accumulation of p53 and p53/47 concomitant with their posttranslational modification, whereas a high dose results in the disappearance of both the forms of p53. The distinct p53 activation profile of LA-12 compared with cisplatin and doxorubicin provides a molecular explanation for the ability of this drug to treat cisplatin-resistant cells and indicates its potential usefulness as an alternative antitumour agent in first-line therapy or as a second-line therapy in patients with acquired cisplatin resistance.