p53: Guardian of reprogramming

The reprogramming of somatic cells to induced pluripotent stem (iPS) cells is one of the major discoveries of recent years. The development and application of patient specific iPS lines could potentially revolutionize cell-based therapy, facilitating the treatment of a wide range of diseases. Despite the numerous technological advancements in the field, an in-depth mechanistical understanding of the pathways involved in reprogramming is still lacking. Several groups have recently provided a mechanistical insight into the role of the p53 tumor suppressor pathway in reprogramming. The repercussions of these findings are profound and reveal an unexpected role of p53 as a "guardian of reprogramming", ensuring genomic integrity during reprogramming at the cost of a reduced efficiency of the process. Here we analyze the latest findings in the field and discuss their relevance for future applications of iPS cell technology.     

Shifting senescence into quiescence by turning up p53

Comment on: Leontieva OV, et al. Cell Cycle 2010; 9:4323–7.     

GLS2 is transcriptionally regulated by p73 and contributes to neuronal differentiation

The amino acid Glutamine is converted into Glutamate by a deamidation reaction catalyzed by the enzyme Glutaminase (GLS). Two isoforms of this enzyme have been described, and the GLS2 isoform is regulated by the tumor suppressor gene p53. Here, we show that the p53 family member TAp73 also drives the expression of GLS2. Specifically, we demonstrate that TAp73 regulates GLS2 during retinoic acid-induced terminal neuronal differentiation of neuroblastoma cells, and overexpression or inhibition of GLS2 modulates neuronal differentiation and intracellular levels of ATP. Moreover, inhibition of GLS activity, by removing Glutamine from the growth medium, impairs in vitro differentiation of cortical neurons. Finally, expression of GLS2 increases during mouse cerebellar development. Although, p73 is dispensable for the in vivo expression of GLS2, TAp73 loss affects GABA and Glutamate levels in cortical neurons. Together, these findings suggest a role for GLS2 acting, at least in part, downstream of p73 in neuronal differentiation and highlight a possible role of p73 in regulating neurotransmitter synthesis.     

Ribosomal protein L37 and the p53 network

Comment on: Llanos S, et al. Cell Cycle 2010; 9:4005–2.     

Skirmish between the mighty p53 protein and the complex retrovirus HIV-1

Comment on: Mukerjee R, et al. Cell Cycle 2010; 9(22):in press.     

Activation of Atg1 kinase in autophagy by regulated phosphorylation

Autophagy is a highly regulated trafficking pathway that leads to selective degradation of cellular constituents such as protein aggregates and excessive and damaged organelles. Atg1 is an essential part of the core autophagic machinery, which triggers induction of autophagy and the Cvt pathway. Although changes in Atg1 phosphorylation and complex formation are thought to regulate its function, the mechanism of Atg1 kinase activation remains unclear. Using a quantitative mass spectrometry approach, we identified 29 phosphorylation sites, of which five are either upregulated or downregulated by rapamycin treatment. Two phosphorylation sites, threonine 226 and serine 230, are evolutionarily conserved and located in the activation loop of the amino terminal kinase domain of Atg1. These phosphorylation events are not required for Atg1 localization to the phagosome assembly site (PAS), or the proper assembly of the multisubunit Atg1 kinase complex and binding to its activator Atg13. However, mutation of either one of these sites results in a loss of Atg1 kinase activity and its function in autophagy and the Cvt pathway. Taken together, our data suggest that phosphorylation of Atg1 on multiple sites provides critical mechanisms to regulate Atg1 function in autophagy and the Cvt pathway.     

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer. Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation. Two p53 homologues, p63 and p73, have been described in humans. Unlike p53, these proteins regulate developmental processes rather than genome stability. Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx. Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined. Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.     

SAC1 encodes a regulated lipid phosphoinositide phosphatase, defects in which can be suppressed by the homologous Inp52p and Inp53p phosphatases

The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity. The activity of Sac1p and its relationship to these phenotypes are unresolved. We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. We have identified two proteins with homology to Sac1p that can suppress drug sensitivity and also restore the levels of the phosphoinositides in sac1 mutants. Overexpression of truncated forms of these suppressor genes confirmed that suppression was due to phosphoinositide phosphatase activity within these proteins. We have now demonstrated this activity for Sac1p and have characterized its specificity. The in vitro phosphatase activity and specificity of Sac1p were not altered by some mutations. Indeed, in vivo mutant Sac1p phosphatase activity also appeared unchanged under conditions in which cells were drug-resistant. However, under different growth conditions, both drug sensitivity and the phosphatase defect were manifest. It is concluded that SAC1 encodes a novel lipid phosphoinositide phosphatase in which specific mutations can cause the sac1 phenotypes by altering the in vivo regulation of the protein rather than by destroying phosphatase activity.