| Background and Objective: Lamin A is a multifunctional protein involved in DNA replication,DNA damage repair,gene expression,transcription regulation,telomere dynamics,and maintenance of chromatin structure.Its phosphorylation modification is an important basis for its movement in the nucleus,the regulation of a variety of protein functions,and promotion of nuclear disassembly during cell division.However,little is known about the regulation of lamin A phosphorylation signal transduction,and the functional significance,as the majority of lamin A phosphorylation sites remain uncharacterized.As well as known that a lamin A G608 G mutation results in the deletion of 50 amino acids at the C-terminus,resulting in the formation of aberrant lamin A(progerin)that accumulates in the nucleus causing cell senescence.Most previous studies focus on progerin cytotoxicity to cells and making nucleus morphological changes,we found that there are multiple phosphorylation sites in the 50 amino acids that are missing in progerin,mass spectrometry analysis found that several of these sites showed high conversion rate characteristics,prompting that they play important roles in cellular functions.The purpose of this study is to search for the functional specific lamin A C-terminal phosphorylation sites and their phosphorylation kinases,and to investigate the effect of site phosphorylation defects on cellular senescence pathways.Methods: We first screened the hyperphosphorylation site of progerin deletion by bioinformatics,and then constructed certain site-mutation plasmids,which were transfected to 293 T cells to express defect proteins.The phosphorylation levels of specific sites of lamin A were analyzed by immunoprecipitation,and key sites were found.Mutations at key sites significantly reduce the overall phosphorylation of lamin A.We then prepared phosphorylation antibodies of these two sites.The kinases which phosphorylated lamin A were confirmed by co-immunoprecipitation and in vitro-kinase incubation assays.In addition,we constructed lamin A mutant cells by a lentivirus expression system,and performed immunofluorescence assays,MTS assays,and ?-gal staining assays to detect the effects on lamin A nuclear localization and cellular senescence pathways.Results:(1)Two important phosphorylation sites of lamin A,Ser628 and Ser636,were identified in this study.It was found that mutations at Ser628 and Ser636 decreased the overall phosphorylation level of laminA protein.(2)We demonstrated that glycogen synthase kinase 3(GSK3?)interacts with lamin A and phosphorylates the lamin A Ser628 site.(3)At the same time,it was found that casein kinase II(CK2)phosphorylates lamin A at Ser636,and it regulates the phosphorylation on lamin A by GSK3? through this site activation.(4)We constructed Ser628 and Ser636 site-mutant cells by lentivirus infection and found that site-defect affects the nuclear distribution of lamin A.(5)In addition,phosphorylation deficient of these two sites affect cell proliferation and cause cell senescence.(6)After DNA damage induction,the mutation cells fail to respond to DNA damage response.Conclusion:Our results demonstrate that GSK3? and CK2 mediate the phosphorylation of lamin A at Ser628 and Ser636.Defection of Ser628 or Ser636 phosphorylation affects nuclear distribution of lamin A and leads to cell senescence.These results suggest that phosphorylation of the C-terminal site of lamin A is involved in the regulation of cellular senescence pathways.Increasing or improving the phosphorylation levels by small-molecule drugs may provide new therapeutic ideas for delaying cell senescence. |
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