Cloning And Functional Study Of Human Gene C4orf13, CDK5RAP1_v3, CDK5RAP1_v4 And PXK

From large-scale human fetal brain cDNA cloning and sequencing plan, we selected four full-length cDNAs by bioinformatics analysis for further research with intention for gene function or related human diseases.The novel human gene (C4orf13) cDNA isolated from fetal brain is 2706bp in length, encoding a 340-amino-acid polypeptide that contains a typical SBF (Sodium Bile acid cotransporter family) domain and ten possible transmembrane segments. The putative protein C4orf13 shows high similarity with its orthologs in Mus musculus and Xenopus laevis. Human C4orf13 is mapped to chromosome 4q31.2 and contains 12 exons. RT-PCR analysis shows that human C4orf13 is widely expressed in human tissues and the expression levels in liver and lung are relatively high, expression levels in placenta, kidney, spleen and thymus are moderate, low levels of expression are detected in heart, prostate and testis. cDNA microarray analysis indicated its expression level is move up in stomach cancer tissue.Two novel splice variants of human CDK5RAP1, named CDK5RAP1_v3 and CDK5RAP1_v4, were isolated. The CDK5RAP1_v3 and CDK5RAP1_v4 cDNAs are 1923bp and 1792bp in length, respectively. Sequence analysis revealed that CDK5RAP1_v4 lacked 1 exon, which was present in CDK5RAP1_v3, with the result that these cDNAs encoded different putative proteins. The deduced proteins were 574 amino acids (designated as CDK5RAP1_v3) and 426 amino acids (CDK5RAP1_v4) in length, and shared the 420 N-terminal amino acids. RT-PCR analysis showed that human CDK5RAP1_v3 was widely expressed in human tissues. The expression level of CDK5RAP1_v3 was relatively high in placenta and lung, whereas low levels of expression were detected in heart, brain, liver, skeletal muscle, pancreas, spleen, thymus, small intestine and peripheral blood leukocytes. In contrast, human CDK5RAP1_v4 was mainly expressed in brain, placenta and testis.Phox homology (PX) domains specifically bind to phosphoinositides,and this interaction is crucial for their cellular function. A full-length cDNA of human PX domain containing serine/threonine kinase gene (PXK) we termed PXKvl had previously been cloned. PXK_vl consists of a STKc domain (serine/threonine kinases, catalytic domain), but lacks several residues that are indispensable for intrinsic catalytic activity. Evidences obtained in present study demonstrated the existence of four other splice isoforms of human PXK in fetal brain, designated as PXK_v2, PXK_v3, PXK_v4 and PXK_v5. The results of RT-PCR indicated human PXKvl, PXK_y2 and PXK_y4 transcripts were widely expressed in human adult tissues except heart. In contrast, PXK_v3 transcripts were only expressed in peripheral blood leukocyte at low level and PXK_v5 transcripts were not detectable in any of the tissues analyzed. The ORF of PXK_vl or PXK_v2 was subcloned into reconstructed pET32a plasmids and the fusion protein was expressed in E.coli Rosette (DE3), and then was purified. No kinase activity of PXK_vl or PXK_v2 has been detected in kinase activity assay. Based on results of sequence analysis and kinase activity assay, it was speculated that PXK proteins might be psudokinases. Subcellular localization analysis of EGFP-PXK fusion proteins in COS7 cells indicated that EGFP-PXK_v3 had a different subcellular localization compared to other EGFP-PXK fusion proteins. Mutation analysis of EGFP-PXKvl showed PXK_vl-Tyr56 and Arg92 are essential for subcellular localization of the protein in the cytoplasm. Over-expression five splice isoforms of human PXK in COS7 cells had effects on cell proliferation at different degrees. In addition, over-expression of five splice isoforms of human PXK could lead COS7 cells to apoptosis. We screened for PXK_vl/PXK_v2-interacting proteins by yeast-two-hybrid system, and no kinases were screened. But we found that protein PPP1R7 can interact with PXK_vl/PXK_v2. It may provided more clue for the next study of the function of PXK proteins.