Functional Studies Of Human Protein Phosphatase The Gene Pp2ck And The Steroid Dehydrogenase Gene Hsdl2

We reported the initial characterization of a cell signal transduction related gene 'PP2Cκ' and a small-chemical-molecule metabolism related gene 'HSDL2'in the two sections of this manuscript.Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells such as cell signal transduction and cell cycle control. A novel member of human protein phosphatases 2C gene named PP2Cκ was isolated from a human fetal brain cDNA library. The 2.0 kb cDNA encodes a 372 amino acid polypeptide with an intact protein phosphatase 2C (PP2C) catalytic domain. The gene is located in human chromosome 4p22 containing 7 exons and 6 introns. Reverse transcription-PCR (RT-PCR) revealed that the PP2Cκ was widely expressed in normal human tissues. The expression levels in adult heart, brain, kidney, pancreas and ovary are relatively high. In eight fetal tissues, PP2C k has higher expression in heart and thymus. Transient transfection suggested that PP2Ck was localized in the nucleus in AD293 cells. Recombinant PP2Ck showed phosphatase activity toward oligopeptides containing phospho-Threonine residues. Furthermore, the overexpression of PP2Ck could distinctly activate the heat shock transcription factor pathway in eukaryotic cells. Our further research indicates that PP2Ck may activate HSF1 pathway by directly interacting with HSF1. PP2Ck and HSF1 co-localized in the cell nucleus and PP2Ck may associate with HSF1 in vitro. This is a new insight to investigate the phosphorylation modifications of HSF1 pathway. We also screen the library by Yeast-two-hybridization system to reveal the candidate proteins which may interact with PP2Ck. Studying the interactins between PP2Ck with those candidate proteins may help us reveal more function information of this widely-expressed gene.Hydroxysteroid dehydrogenases (HSDs) are responsible for the biosynthesis and metabolisms of steroid hormones and play a crucial role in mammalian physiology and development. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human hydroxysteroid dehydrogenase like cDNA (HSDL2). This cDNA is 3211bp in length, encoding a 418-amino-acid polypeptide which contains an intact dehydrogenase catalytic domain and a sterol carrier proteindomain. A conserved cofactor NAD(P)(H) binding motif TGxxxGxG and SDR active site YxxxK were also found in this protein. The HSDL2 shows high similarity with the homologues in the mouse and fruit fly. The HSDL2 gene is mapped to human chromosome 9q32 and contains 11 exons. RT-PCR analysis shows that the HSDL2 gene is widely expressed in human tissues and the expression levers in liver, kidney, prostate, testis, and ovary are relatively high. Bacterial expressed recombinant HSDL2 protein has the capacity of binding NADPH. By using the green fluorescent protein (GFP) marker, we investigate that HSDL2 protein localizes in the cytoplasm. We also found that the cell total sterol capacities remarkably increased when overexpressing the SCP-2 domain containing protein-HSDL2. This suggest the potential roles of HSDL2 in sterol metabolisms.