Association of Tat with Promoters of PTEN and PP2A Subunits Is Key to Transcriptional Activation of Apoptotic Pathways in HIV-Infected CD4+ T Cells

Apoptosis in HIV-1-infected CD4+ primary T cells is triggered by the alteration of the PI3K and p53 pathways, which converge on the FOXO3a transcriptional activator. Tat alone can cause activation of FOXO3a and of its proapoptotic target genes. To understand how Tat affects this pathway, we carried out ChIP-Chip experiments with Tat. Tat associates with the promoters of PTEN and two PP2A subunit genes, but not with the FOXO3a promoter. PTEN and PP2A encode phosphatases, whose levels and activity are increased when Tat is expressed. They counteract phosphorylation of Akt1 and FOXO3a, and so activate transcriptional activity of FOXO3a. FOXO3a promotes increased transcription of Egr-1, which can further stimulate the transcription of PTEN, thereby reinforcing the pathway that leads to FOXO3a transcriptional activation. RNAi experiments support the role of PTEN and PP2A in the initiation of the Tat-mediated cascade, which is critical to apoptosis. The increased accumulation of PTEN and PP2A subunit mRNAs during Tat expression is more likely to be the result of increased transcription initiation and not relief of promoter-proximal pausing of RNAPII. The Tat-PTEN and -PP2A promoter interactions provide a mechanistic explanation of Tat-mediated apoptosis in CD4+ T cells

A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat

Background: We aimed to further understand the relationship between cAMP concentration and mnesic performance. Methods and Findings: Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/ kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (,0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h)

Mechanism of activation of PKB/Akt by the protein phosphatase inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels. © 2010 Springer Science+Business Media, LLC.We thank the European Community programme ‘‘Quality of Life and Management of Living Resources’’ for a Marie Curie Fellowship (QLK1-CT-2000-51184), ‘‘Ministerio de Educación y Ciencia’’ grant BFU2006-01088/BMC and ‘‘Programa Ramón y Cajal’’ contract (B.O.E. 17/02/2004 ORDEN CTE/351/2004) to Mercedes Pozuelo Rubio.Peer Reviewe