Human Rpk118 Gene Function Study

PRS6KC1 was reported by our research group in 1999, and its expression was up-regulated in hepatocellular carcinomas. After further analysis of sequence, PRS6KC1 turned out to be incomplete. Through screening the human EST database of GenBank, some new ESTs of 5'-terminal region were obtained, and the full-length sequence of RPS6KCI cDNA was determined. In consistent with the transcript of about 4.4kb showed in Northern blot hybridization, this cDNA has 4196bp, and 5'-terminal of the cDNA was 1437 bp longer than previous sequence (2349bp). It contains an open reading frame consisting 320lbp that encodes a putative protein of 1066 amino acids with a calculated molecular mass of 118.7 kDa. The new sequence of RPS6KC1 cDNA was used as probe to screen the human Genome database, it showed to have 15 extrons and 14 introns and located in 1q32.3. We cloned and identified the full-length PRS6KC1 cDNA from the human brain cDNA library. By using the SMART algorithm, we had identified four domains homologous to previously characterized proteins including a phox homology (PX) domain, a MIT (Microtubule -Interacting and Trafficking moleculars)domain, and STK1 and STK2 domain ( Serine/ Threonine protein kinases, catalytic domain) .We produced and purified PRS6KC1 polyclonal antibodies and identified PRS6KC1 as a 118kDa protein in cells. The immunoprecipitated 118kDa-protein showed no kinase activity toward the exogenous substrates such as MBP(myline binding protein) and S6 peptide, suggesting that the protein is defective in phosphotansferase activity, it was the same as the result of the Japanese researcher(JBC. 2002.9). Thus, we accepted RPK118(Ribosomal S6 kinase-like protein with two PseudoKinase domains) instead of RPS6KC1 as the name of this gene.The interacting proteins of RPK118 were screened through Yeast two-hybrid system. Peroxiredoxin-3(PRDX3) is found to be one of RPK118-binding proteins, The interaction between RPK118 and PRDX3 is proved by further immunoprecipitation and GST pull-down studies. Similarly to SPHKl(sphingosine kinase), which was reported recently, PRDX3 was also shown to co-localize with RPK118 on early endosomes in COS7 and 293T cells. RPK118 was distributed diffusely in the cytosol and in some small dot-like or ring-shaped structures. When EEA1, an endogenous marker of early endosome was immunostained, RPK118 displayed good co-localization with EEA1, indicating that the RPK118-potitive dot-like or ring-shaped structures were in fact early endosomes. When PRDX3 and RPK118 were expressed simultaneously in COS7 cells, RPK118 was distributed as a similar pattern as this protein was...