Characterization Of Acidic Ribosomal Phorphosrylated Proteins From Euplotes Octocarinatus

Eukaryotic ribosomal P0, P1, and P2 proteins (P proteins) and the conserved domain of 28S rRNA constitute a major part of the GTPase-associated center in eukaryotic ribosomes. They form a pentameric complex and play a crucial role in interaction with translation factors. These proteins share a conserved flexible C-terminal tail that is rich in acidic residues, which are involved in the interaction with elongation factor during protein synthesis. The last 11 residues of these proteins, SDD/EDMGFGLFD, are highly conserved among majority of eukaryotic acidic ribosomal proteins. Several specific properties are observed with eukaryotic P proteins. First, these proteins exist on the ribosome as phosphorylated derivatives. They can be phosphorylated in vitro by either casein kinase 2 or by an endogenous ribosome-bound enzyme. Second, phosphorylation of P1 and P2, which appears to be necessary for ribosome activity, was originally suggested to be a requirement for the binding of these proteins to ribosomes. Third, phosphorylation of P proteins is beneficial for their participation in the elongation process of protein synthesis. CK2 is a ubiquitous protein kinase responsible for the phosphorylation of Ser and Thr residues specified by acidic side chains in many proteins, including many key enzymes, growth factor receptors, transcription factors and cytoskeletal proteins. The CK2 holoenzyme is composed of two catalytic (a and a’) and two regulatory subunits (β).Like other ciliates, Euplotes octocariuatus has two types of nuclei:micronucleus and macronucleus. The micronucleus contains conventional chromosomes, whereas the DNA in the macronucleus is organized as short, linear molecules. This characteristic makes it possible to clone the gene from Euplotes octocariuatus using a telomeric primer and an internal gene specific primer. Another striking characteristic of Euplotes is its deviation from the universal genetic code. The codon TGA is translated into cysteine instead of functioning as a termination signal. It has been demonstrated that more than 5% of genes in Euplotes undergoes programmed translational frameshift, most of which was+1 frameshift.In this study, genomic DNA and total RNA were isolated from Euplotes octocarinatus, and genes for acidic ribosomal protein P0, P1 and P2 genes (named EoPO, EoPl and EoP2, respectively) were cloned by PCR strategies. Recombinant plasmids pRSETC-EoPO and pRSETC-EoP2 were successfully constructed, the recombinant proteins His-EoPO and His-EoP2 were expressed in E. coli BL21 and purified by Ni-NTA Sepharose affinity chromatography. EoP2 phosphorylation was studied using NMR, native PAGE and Phos-tagTM SDS-PAGE. Below is a summary of key results.1. Cloning of P0, P1 and P2 genes from Euplotes octocarinatus. The coding sequence is 1002 bp for EoPO gene is and 336 bp for EoP2 gene. Bioinformation analysis and PCR sequencing showed that the sequence of coding region of EoPl gene is 354 bp. These sequence data clearly show that the C-terminal regions of P proteins from E. octocarinatus are very different from those in other eukaryotes. The C-teriminal domains of EoPO and EoP2 are 5-7aa longer than that observed in other eucaryotics. On the other hand, the C-teriminal domain of EoP1 is 5-7aa shorter than that observed in other eucaryotics. These contrast results showed that EoPO, EoPl and EoP2 have low homology.2. Expression of fusion protein His-EoPO and His-EoP2. EoPO and EoP2 were cloned into expression vector pRSETC and expressed in E. coli BL21. His-EoP2 was purified and exchanged medium by Ni-NTA Sepharose affinity chromatography and size exclusion column. Expression of each recombinant protein was detected by 15% SDS-PAGE and confirmed by Western blotting. Mice were immunized with purified recombinant EoP2 to generate specific polyclonal antibody.3. Phosphorylation detection of EoP2. Seven potential phosphorylation sites of EoP2 were predicted using software NetPhos 2.0:Ser19 (0.991), Ser21 (0.991), Ser29 (0.661), Ser66 (0.868), Thr17 (0.665), Thr41 (0.958) and Tyr112 (0.960); Phos-tag SDS-PAGE and NMR showed that EoP2 was phosphorylated by CK2. The predicted high score of EoP2 phosphorylation sites Ser19 (0.991), Ser21 (0.991), Thr41 (0.958) and Tyr112 (0.960) were mutated using site-directed mutagenesis method. Each of these protein mutants was expressed and purified. NMR results showed that WT-EoP2, EoP2-S19A, EoP2-T41A, EoP2-Y112A were phosphorylated in vitro by CK2 except EoP2-S21A and EoP2-S19A-S21A, suggesting that EoP2 was phosphorylated in vitro at S21. Additionally,, the phosphorylation of EoP2 was also analyzed by native PAGE. Phosphorylation led to the appearance of a distinct band with higher mobility than that of free EoP2.4. Assay of interaction between EoP2 and EoEF-2. Elongation factor 2 genes of Euplotes octocarinatus (EoEF-2) was cloned by PCR; TGA in EoEF-2 gene was point mutated to TGT to express EoEF-2 in vitro; six mutatants of EoEF-2 and EoP2 were successfully expressed and purified. Interaction between EoP2 and EoEF-2 was studied with a pull-down method, and results showed that the interaction site is located at the C-terminal domain of EoP2 and the N-terminal domain of EoEF-2. To simulate the phosphorylation of EoP2, we mutated the site of S21 to D21. The results showed that phosphorylation of EoP2 didn’t improve interaction between EoEF-2 and EoP2-S21D.To conclude, we cloned the acidic ribosomal phosphorylated protein P0, P1 and P2 genes from Euplotes octocarinatus. Sequence analysis showed that the C-terminal regions of P proteins from E. octocarinatus are very different from those in other eukaryotes. Interaction between EoP2 and EoEF-2 was detected by pull-down via the C-terminal domain of EoP2 and the N-terminal domain of EoEF-2. It is suggested EoP2 may play an important role in the elongation process of protein synthesis. The analysis of phosphorylation sites of EoP2 showed that position S21 was phosphorylated in vitro, which is also quite different from that in other organisms. However, the phosphorylation of EoP2 didn’t affect the interaction between EoP2 and EoEF-2, suggesting that phosphorylation of EoP2 had peculiar biological function in the elongation process of protein synthesis. The current study on characterization and function of EoP2 phosphorylation opens a new way to further understand the regulatary role of ribosomal proteins in protein synthesis in lower eukaryotes.