Study On The Mechanism Of PP2A-B56γ Participating In RAS Induced Cell Senescence Through KSR1

In recent years,oncogene induced senescence(OIS)has attracted extensive attention of researchers.OIS can limit the rapid proliferation of abnormal cells and prevent the occurrence and development of tumors.It is a natural barrier for human body to prevent tumors.Under normal circumstances,the over activation of RAS will trigger OIS and inhibit the occurrence of tumor.However,if OIS goes wrong,over activated RAS will lead to unlimited cell division and eventually lead to cancer.Protein phosphatase 2A(PP2A)is one of the four major serine/threonine phosphatases,which is involved in cell division,growth and other functions.Various variants or abnormal expression of PP2 A often occur in cancer.Previous studies have shown that SV40 ST inhibited RAS induced senescence(RIS)by interfering with the function of PP2A-B56γ,but its specific mechanism is unknown.In this study,we first used WB,IF and SA-β-Gal staining proved that the action link of SV40 st should be between BRAF and MEK.Then,known through the literature that RAS kinase inhibitor of RAS 1(KSR1)was found to play an important role in Raf signaling pathway as a scaffold protein in recent years,and is related to cancer rest.KSR1 is located between BRAF and MEK signal pathway and is related to PP2 A.Next,we knocked down KSR1 in RAS overexpressed cells and performed IF and SA-β-Gal staining showed that after knockdown of KSR1,the aggregation of cell DNA damage decreased and the proportion of senescent cells decreased,which proved that knockdown of KSR1 inhibited RAS induced cell senescence.WB detection showed that knockdown KSR1 inhibited the phosphorylation of ERK1/2,thus inhibiting cell aging.Then we overexpressed KSR1 in cells.Through IF and SA-β-gal detection,it was found that overexpression of KSR1 alone did not induce cell aging.WB experiment also proved that the phosphorylation level of ERK1/2 did not increase significantly.It can be seen that simple overexpression of KSR1 will not cause cell aging.KSR1 knockdown can inhibit RAS induced cell senescence,while KSR1 overexpression cannot induce cell senescence.In order to further confirm the role of KSR1 in RIS,we performed KSR1 compensation in cells with RAS overexpression and knockdown of KSR1.The results showed that the DNA damage of cells after KSR1 compensation increased and the proportion of aging cells increased.The WB experiment showed that the phosphorylation of ERK1/2 increased.The above results show that KSR1 is indeed involved in RAS induced cell senescence.After that,we detected the WB of RAS overexpressing cells and found that the expression of PP2A-B56γ was up-regulated and the expression of p-KSR1 was down regulated.WB detection of overexpressing PP2A-B56γ showed that the expression of p-KSR1 decreased,and enhanced EGF induced cell senescence.Knockout of PP2AB56γ in RAS overexpressing cells showed that the level of p-KSR1 was up-regulated.Thus,PP2A-B56γ acts on RAS signaling pathway by regulating the phosphorylation level of KSR1.Based on the above research results and literature information,we conclude that PP2A-B56γ maintains the continuous activation of RAS signal pathway by regulating the phosphorylation of KSR1,and then affects the process of RIS;SV40 ST inhibits RIS by interfering with the normal function of PP2A-B56γ and inhibiting the over activation of RAS.