p14ARF activates a Tip60-dependent and p53-independent ATM/ATR/CHK pathway in response to genotoxic stress.

p14ARF is a tumor suppressor that controls a well-described p53/Mdm2-dependent checkpoint in response to oncogenic signals. Here, new insights into the tumor-suppressive function of p14ARF are provided. We previously showed that p14ARF can induce a p53-independent G2 cell cycle arrest. In this study, we demonstrate that the activation of ATM/ATR/CHK signaling pathways contributes to this G2 checkpoint and highlight the interrelated roles of p14ARF and the Tip60 protein in the initiation of this DNA damage-signaling cascade. We show that Tip60 is a new direct p14ARF binding partner and that its expression is upregulated and required for ATM/CHK2 activation in response to p14ARF. Strikingly, both p14ARF and Tip60 products accumulate following a cell treatment with alkylating agents and are absolutely required for ATM/CHK2 activation in this setting. Moreover, and consistent with p14ARF being a determinant of CHK2 phosphorylation in lung carcinogenesis, a strong correlation between p14ARF and phospho-CHK2 (Thr68) protein expression is observed in human lung tumors (P < 0.00006). Overall, these data point to a novel regulatory pathway that mediates the p53-independent negative-cell-growth control of p14ARF. Inactivation of this pathway is likely to contribute to lung carcinogenesis.

p14ARF triggers G2 arrest through ERK-mediated Cdc25C phosphorylation, ubiquitination and proteasomal degradation.

The Cdc25C phosphatase is a key regulator of mitotic entry which activity is tightly regulated by phosphorylation. In response to DNA damage, phosphorylation at serine 216 induces the cytosolic retention of Cdc25C through 14-3-3 binding. We previously reported the ability of the p14ARF tumor suppressor to induce the accumulation of inactive phospho-Cdc25C(Ser216) protein as well as a decrease of Cdc25C steady state level and correlated these events with a p53-independent G2 arrest. The aim of this study was to investigate the cellular signaling pathways involved in this process. By using specific pharmacological inhibitors, we demonstrate that activation of the ERK1/2 MAP kinases pathway is involved in the p53-independent G2 checkpoint induced by p14ARF Moreover, we show that activated P-ERK1/2 bind and phosphorylate Cdc25C on its ser216 residue following p14ARF expression, thereby identifying Cdc25C as a new ERK1/2 target. Importantly, we further show that phosphorylation at Ser216 by phospho-ERK1/2 promotes Cdc25C ubiquitination and proteasomal degradation, suggesting that Cdc25C proteolysis is required for a sustained G2 arrest in response to p14ARF. Taken together, these results demonstrate that the MAPK ERK signaling pathway contributes to the p53-independent antiproliferative functions of p14ARF. Furthermore, they identify a new mechanism by which phosphorylation at serine 216 participates to Cdc25C inactivation.

The precursor of a psychrophilic alpha-amylase: structural characterization and insights into cold adaptation.

The alpha-amylase precursor from the bacterium Pseudoalteromonas haloplanktis possesses a propeptide at the C-terminus possibly responsible for outer membrane translocation. Unlike the predicted beta-barrel of autotransporters, this C-terminal propeptide displays a noticeable alpha-helix content. It is connected to the enzyme by a disordered linker and has no significant interaction with the catalytic domain. The microcalorimetric pattern of the precursor also demonstrates that the stability of protein domains may evolve differently.

Molecular basis of cold adaptation.

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.