Identification of HN252 as a potent inhibitor of protein phosphatase PPM1B.

Protein phosphatase 1B (PPM1B), a member of metal-dependent protein serine/threonine phosphatase family, is involved in the regulation of several signalling pathways. However, our understanding of its substrate interaction and physiological functions is still largely limited. There is no reported PPM1B inhibitor to date. In this study, we identified HN252, a p-terphenyl derivative, as a potent PPM1B inhibitor (Ki  = 0.52 ± 0.06 µM). HN252 binding to PPM1B displayed remarkable and specific inhibition of PPM1B in both in vitro and ex vivo. With the aid of this small molecular inhibitor, we identified 30 proteins' serine/threonine phosphorylation as potential substrates of PPM1B, 5 of which were demonstrated by immunoprecipitation, including one known (CDK2) and 4 novel ones (AKT1, HSP90B, ß-catenin and BRCA1). Furthermore, GO and KEGG analysis of dramatically phosphorylated proteins by PPM1B inhibition indicated that PPM1B plays roles in the regulation of multiple cellular processes and signalling pathways, such as gene transcription, inflammatory regulation, ageing and tumorigenesis. Our work provides novel insights into further investigation of molecular mechanisms of PPM1B.

Isolation of a novel protein phosphatase2C in rice and its response to gibberellin.

Protein phosphatase 2Cs (PP2Cs) have been referred to act as negative modulators of the protein kinase pathways involved in different environmental stress responses and developmental processes. In Arabidopsis, PP2Cs have been extensively studied and some are known to negatively regulate abscisic acid signaling. In rice, PP2Cs are scarcely characterized functionally. Here, we identified a novel PP2C from rice (OsPP2C34), which is highly inducible by gibberellin (GA) and expressed in various tissues. Subcellular localization analysis in maize protoplasts using a green fluorescence protein fusion vector localized OsPP2C34 to the cytosol. Genetic analysis of T-DNA insertional mutants revealed that plant height and internode length were significantly shorter in mutants than in corresponding wild types under GA treatment. The induction of the GA-inducibleα-amylase genes RAmy3E and OsAmy was delayed in mutant plants. The substrate of OsPP2C34 was identified by immunoblotting using anti serine/threonine antibodies. A 65 kDa protein was phosphorylated in Ospp2c34-1 but dephosphorylated in the wild type during early germination stage. Overall, the present results indicated that OsPP2C34 is involved inα-amylase expression of GA signal transduction pathway.

Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum.

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H2O2, ZnCl2, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.