PKMYT1 Inhibits Phosphorylation Of AKT And Suppresses NSCLC Proliferation

BackgroundWorldwide,lung cancer represents a main cause of cancer-related deaths.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer,accounting for approximately 85%of total lung cancer cases.As NSCLC is highly invasive without apparent symptoms at early stages and early diagnostic biomarkers,most patients are clinically diagnosed at advanced stages,and the 5-year survival rate remains low.Therefore,it is urgent to identify key molecules that regulate the malignant phenotype of NSCLC and explore effective therapeutic targets as well as diagnostic markers.Serine/threonine kinase AKT is critical for the survival and proliferation of tumor cells and aberantly activated in many tumor types.Previous studies showed that approximately 51%NSCLC possess increased AKT activity,mostly presented as elevated AKT phosphorylation and overactivation of its downstream signaling pathways,which is also closely related to the poor prognosis of NSCLC patients.Significantly,investigating the specific molecular events key to AKT phosphorylation may provide an important basis for the development of new drugs targeting AKT activation and blocking AKT signaling.Based on the above research background,combined with previous results of mass spectrometric analysis of AKT immunoprecipitated proteins carried out in our laboratory,we hypothesized that PKMYT1 might be a potential kinase that interacts with AKT in NSCLC.In such a context,the following scientific questions are addressed in this thesis:Does PKMYT1 bind AKT?What are the biological effects of the interaction between PKMYT1 and AKT?What are the biological effects of PKMYT1 on NSCLC?Methods1.Study on the interaction between PKMYT1 and AKT(1)Immunoprecipitation assay(Co-IP)and far-Western Blotting assay were used to verify the binding between PKMYT1 and AKT.(2)Plasmids expressing truncated PKMYT1 and AKT were constructed,and the binding domains of PKMYT1 and AKT were identified by co-IP assay.(3)Western blotting assay was used to detect the effects of overexpression or knockdown of PKMYT1 on the phosphorylation levels of AKT T308 and S473.(4)Co-IP assay was employed to examine the effect of PKMYT1 on the binding of PDK1 or ILK1 with AKT.2.Study on the effect of PKMYT1 on the proliferation of NSCLC cells.(1)The effects of overexpression or knockdown of PKMYT1 on the proliferation of NSCLC cells in vitro were detected by plate clony formation assay,Thiazolyl blue cell growth assay(MTT)and Soft Agar clone formation assay(Soft Agar).(2)The effect of overexpression or knockdown of PKMYT1 on tumorigenicity of NSCLC cells in nude mice was detected by subcutaneously injected xenografts tumorigenicity assay in nude mice.(3)Plate clone assay,MTT assay,Soft Agar assay and EdU assay were used to detect the effects of PKMYT1 kinase domain mutation on NSCLC cell proliferation.Results1.PKMYT1 binds AKT and down-regulates the phosphorylation level of AKT.(1)PKMYT1 can directly bind AKT.(2)The kinase domain of PKMYT1 interacts with the catalytic domain of AKT.(3)Excessive expression of PKMYT1 in NSCLC cells decreases the phosphorylation level of AKT T308 and S473.(4)PKMYT1 binding to AKT competitively abrogates the binding of AKT with PDK1 and ILK1.2.PKMYT1 inhibits the proliferation of NSCLC.(1)PKMYT1 inhibits the proliferation of NSCLC cells in vitro and in vivo.(2)PKMYT1 mutant(N238A,D251A)reverses the inhibition of NSCLC cell proliferation.Conclusions1.PKMYT1 binds AKT directly,and the kinase domain of PKMYT1 interacts with the catalytic domain of AKT.2.PKMYT1 inhibits AKT phosphorylation by competitively antagonizing AKT phosphorylation induced by PDK1 and ILK1.3.PKMYT1 suppresses the proliferation of NSCLC cells.4.PKMYT1 N238 and D251 contribute to the inhibition of NSCLC proliferation.