Study On Protein Tyrosine Phosphotases And Protein Tyrosine Kinases Correlated With Diseases

Protein phosphorylation is controlled by the coordinate action of proteinkinases and phosphatases and has a crucial role in cell proliferation,differentiation, and transformation. Protein tyrosine kinases are centralregulators of signaling pathways. Perturbation of PTK signaling bymutations and other genetic alterations such as chromosomal translocation,interstitial deletion, internal tandem duplication, and amino acid substitutionresults in deregulated kinase activity and malignant transformation.The mutation of PTKs are correlated with the tumor or cancer in human,so many research groups focus on the relation between PTKs and humandiseaseJanus kinases (Jaks) are non-receptor tyrosine kinases, In mammals, thefamily has four members, Jak1, Jak2, Jak3 and Tyk2 and play an important rolein the cellular signal transduction of cytokine receptor superfamily, becausethere are not the tyrosine kinase domaim of the cytokine receptor and so thetyrosine phosphorylation controlled by JAK is more important. Jak2 deficiencyresults in embryonic lethality at embryonic day 12.5 as a result of a failure indefinitive erythropoiesis, so JAK2 has a crucial role in hematopoiesis.JAK/ STAT(Signal Transducer and Activators of Transcription ) pathwayhas been extensively studied and the important role has been demonstrated inimmune system regulation, cell proliferation, anti-apoptosis , cell cycleregulation and tumor cell growth. The cell proliferation and differentiation ofhematopoietic cells are controlled by many cytokines including G-CSF, EPO,TPO, IFN and IL. The cytokines combined with their receptor and induced thetyrosine phosphorylation by JAK. The abnormal cellular signal transduction ofJAK /STAT pathway can induce abnormal hematopoiesis。Polycythemia vera (PV) is a clonal myeloproliferative disorder (MPD)characterized by trilineage marrow hyperplasia with increased production of redblood cells, granulocytes, and platelets. PV mostly occurrences in middle andold age population and males are more than females. The process of PV isslowly and patients coincidence with thrombus and embolism, sometimesdeveloped acute leukaemia. Lacking effective therapeutic tool, the mortality ofPV is very high. Therefore the early diagnosis of PV is more important.Between March and June 2005, five studies described a novel JAK2somatic point mutation (a G-C to T-A transversion, at nucleotide 1849 of exon14, resulting in the substitution of valine to phenylalanine at codon 617 in about90% PV patients. The findings defined the molecular defection in PV. The JH2domain can inhibit the activation of JAK2 kinase, and the mutation induced thedecreasing inhibition role and enhancing the activation of kinase. The mutationis acquired, and some mutant is heterozygous.Subsequently, the same mutation was demonstrated in other myeloiddisorders including acute myeloid leukemia, the myelodysplastic syndrome,chronic myelomonocytic leukemia, juvenile myelomonocytic, leukemia, chronicneutrophilic leukemia, hypereosinophilic syndrome, systemic mastocytosis(SM), and other atypical MPD, the occurrence of mutation is less than PV.In order to define the correlation between JAK2V617F and MPDs, weprepared genomic DNA from a total of 3013 peripheral blood samplesrandomly collected from the blood test laboratory of China Japan UnionHospital, Jilin University. We relied on the allele-specific PCR method toanalyze genomic DNA from blood samples;we found 34 positive samples outof a total of 3013, corresponding to a percentage of 1.1%.To further verify the results obtained from allele-specific PCR assays,we sequenced the 521 bp PCR fragment obtained from PCR positive samplesin the initial PCR. Sequencing results confirmed 9 of the samples whichcontained at least 7% of the mutant DNA. These samples corresponded torelatively higher amounts of the mutation specific PCR products. Theremaining 25 samples contained less than 5% of mutation and could not beunambiguously confirmed by DNA sequencing. Our study thus revealed anunexpectedly high rate of the JAK2V617F mutation in bloods.The statistics for blood test data of the entire samples displayed themean of WBC and platelet of JAK2 mutant group are more than the wholetest group, it maybe imply the JAK2V617F play an important role in theregulation of hematopoiesis. In our study, it is of significance for earlydiagnosis, progonosis and adaptive therapy of PV.Protein phosphorylation is controlled by the coordinate action of proteinkinases and phosphatases and involves in many human diseases such asdiabetes, leukaemia, cancers and immunodeficiency, therefore studying onPTPs has been more and more important.PRL is a kind of endocellular enzyme, consisting of PRL-1, PRL-2 andPRL-3, and their amino acid sequence similarity is above 75%. The molecularweight of PRL is about 20KDa. PRLs play a crucial role in cell proliferation,differentiation, and transformation.The abnormal expression of PRLs can influence the proliferation andinvasive growth of non-tumor cells.Employing siRNA interference theexpression of the PRL-1 and PRL-3 in murine tumor cell displayed thedecrease of the proliferaton and migration of the tumor cells.Recent study shows PRL phosphatases increase cell proliferation bystimulating progression from G1 into S phase. PRL-1 function is regulated ina cell cycle-dependent manner and implicates PRL-1 in regulatingprogression through mitosis, possibly by modulating spindle dynamicsPRL-3 expression in colorectal cancers may contribute to theestablishment of liver metastasis, particularly at the step in which cancer cellsleave the circulation to extravasate into the liver tissue. In addition, PRL-3 isexpected to be a promising biomarker for identifying colorectal cancer patientsat high risk for distant metastasesRNA interference (RNAi) has undergone a rapid development as apowerful method to the research of Gene Therapy and the Functional Genomicssince its discovery by Fire etc. in 1998. It triggers different types of genesilencing by recognition and ultimately the cleavage or translational repressionof complementary single-stranded RNAs such as mRNA, and leads topost-transcriptional gene silencing.C.elegans is a perfect RNAi carrier with short lifespan. It is easy to feedand its transparent epidermis can ease the process of observation. Moreimportant, it is a eucaryotic metazoan whose cells, molecular constructions andregulating pathways are correspondent with higher animals. All these aboveunique advantages make C.elegans an excellent model organism.Now, it is widespread abroad to use RNA interference (RNAi) andC.elegans as model organism. Surprisingly, there are still blanks in China.Therefore, my dissertation is to establish a set of systematic methods of RNAiwith C.elegans as model organisms in order to provide some developed methodsfor the investigation on the construction and function of organism.The main procedures of my research are as follows:Firstly, we design primers, obtain the cPRL gene from the cDNA databaseby use of PCR, construct to clone vector (pBluescript II KS) and expressionvector (pGex-2T), transform the later vector to appropriate E.coli (DE3), selectpositive clone, and induce with IPTG.. The result of SDS-PAGE shows that wegain highly expressed GST-cPRL fusion protein .Secondly, we purify the GST-cPRL fusion protein with GSH-Sephorose 4B.After preparation of purified protein, we start immunoreactions in rabbit and getthe serum of cPRL of which the titer is about 1:10000. To increase the specialcombination to antigen, the antibody is then purified using affinitychromatography and PVDF immobilized antigen affinity chromatography. Thefinal titer is about 1:2000.Finally, we analyze the biological functions of cPRL gene by RNAi.Theresults of immunohisto-chemistry and immunofluorescence show that cPRLmainly expresses in the intestine lateral wall, while the analysis of Westernblotting indicates cPRL expresses in larvae stage, which implicates the probableinvolvement of cPRL in intestine development.From the comparison of the physiological appearances between RNAigroups and Control groups, we find that the ability of dynamics of RNAi groupsis more powerful than that of Control groups at the same life stage, includingincreased move rate and decreased defecation time interval which are necessaryphysiological functions for life. In addition, the lifespan of RNAi groups issignificantly extended. We can make a conclusion from all the findings thatcPRL plays a crucial role in the regulation of C.elegans lifespan.Through the above experiments, we establish a systematic set of methods instudying the enzymes' biological functions by RNAi technology usingC.elegans as a model system, such as obtaining, cloning and expressing thespecific enzyme gene of C.elegans, isolating and purifying the enzyme,preparing the polyclonal antibody, RNA interfering of target gene, andobserving the phenotypes after interfering, all of which there are no relatednation-wide reports. Additionally, we primarily explore the biological functionof cPRL gene by interfering C.elegans and analyzing the phenotypes after RNAi.We expect that our study on C.elegans PRL gene will contribute to furtherinvestigation into the mechanism of cPRL and provide an interrelated theory forthe drug selection and therapy of PRL related human diseases.