In Addition To Research And Molecular Studies In Mouse Models EGFR Phosphorylation Mechanism Zkscan3 Turn Knockout

ObjectivePrevious studies from our group originally focusing on colorectal carcinoma,prostate cancer, and multiple myeloma determined that zinc finger with KRAB andSCAN domains3(zkscan3), a novel zinc-finger transcription factor, coordinated a geneexpression program and was an important driver for tumor development, migration, andpathogenesis, as well as autophagy regulation. However, the biological role of zkscan3gene under normal physiological condition remains largely unknown. We thereforeestablished a zkscan3gene knock-out mouse model which has not been reported yet, tosystematically analyze the phenotype and finally understand the major physiologicalrole of zkscan3gene.Methods1. The exon2of zkscan3gene will be knockout (KO) by CRE hybrid system andgenotyping will be performed to identify offspring.2. Semi-quantitative RT-PCR will be performed to examine the expression ofzkscan3genes in various organs of wild-type mice.3. Quantitative PCR (qPCR) will be also performed to verify the zkscan3geneknockout efficacy in mouse embryonic fibroblasts and several representative organs.4. Flow cytometry will be used to analyze the difference of CD3+cells in thymusand CD3+, CD4+, CD8+cells in spleen between adult wild-type and zkscan3knockoutmice.5. Mouse embryonic fibroblasts of zkscan3knockout and wild type mice will beanalyzed by cDNA microarray to identify the genes that potentially modulated byzkscan3gene.6. The immune response of mouse models will be examined by CFA immunizationand detected by determining the immune system response curve of IgG and IgM. Results1. Knockout of zkscan3gene exon2was demonstrated by genotyping.2. Expression of zkscan3gene in various organs of mice was determined byRT-PCR. The results showed that zkscan3mRNA is expressed in different organs withvaried expression levels.3. qPCR results showed that the2nd exon of zkscan3gene was indeed knockout.High expression levels of zkscan3genes in stomach, ovary, spleen and prostate ofwild-type mice were observed as well by qPCR.4. Flow cytometry analysis indicated that CD3+lymphocytes in the KO thymushas significant more percentage than in wild-type mice, while percentages of CD4+andCD8+lymphocytes in the knockout mice spleen significantly increase than those inwild-type.5. Microarray results show that deficiency of zkscan3gene results in significantchanges of genes those mainly affect the nervous, immune, and reproductive systems.6. By immunization of CFA, we found that immune response in KO mice appearsdisorganized response curves, meanwhile blood tests index shows that zkscan3knockout mice may have symptoms of anemia.ConclusionPreliminary results show that zkscan3knockout mice exhibits increased immunefunction, and may also play a role in nervous and reproductive system. ObjectiveEpidermal growth factor receptor (EGFR), a well-known oncoprotein, plays animportant role in cell growth, cell cycle, and many cancerous diseases. Althoughdimerization of EGFR has been documented as a major process for its activation, it remains elusive as to how EGFR is activated afterwards and how itstransphosphorylation and autophosphorylation interact with each other. Here, wedemonstrated that transphosphorylation of EGFR at Y845plays an essential role on itsautophosphorylation as well as kinase activity. This observation may provide newinsights into the mechanisms of EGFR activation and functional regulation.Methods1. Immunoprecipitation and immunoblot.2. In vitro kinase assay.3. For in vitro transphosphorylation assay.4. Transfection and establishment of stably expressing EGFR cell clones.5. Cell growth assay.6. In vitro DNA incorporation assay.Results1. Transphosphorylation of EGFR at Y845affects its autophosphorylation.2. The transphosphorylation deficient EGFR-Y845F mutant abrogates its kinaseactivity.3. The transphosphorylation deficient EGFR-Y845F mutant abrogates its abilityto phosphorylate its dimeric partners.4. The EGFR-Y845F mutant reduces its ability to stimulate cell growth and DNAsynthesis.ConclusionTransphosphorylation of EGFR at Y845is linked to its autophosphorylation andkinase activity. The substitution of Y845to phenylalanine abrogated its biologicalfunction in response to ligand stimulation, suggesting the importance of thistransphosphorylation site. Taken together, our results provide insights into the activationof EGFR and may indicate a potential therapeutic target for treating various cancersrelated to aberrant EFGR expression and activation.