title: Identification of the lamin A/C phosphoepitope recognized by the antibody P-STM in mitotic HeLa S3 cells.
abstract: BACKGROUND
  Lamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. Phosphorylation of lamin A/C is cell cycle-dependent and is involved in regulating the assembly-disassembly of lamin filaments during mitosis. We previously reported that P-STM, a phosphoepitope-specific antibody raised against the autophosphorylation site of p21-activated kinase 2, recognizes a number of phosphoproteins, including lamins A and C, in mitotic HeLa cells.
  
  
  RESULTS
  Here, using recombinant proteins and synthetic phosphopeptides containing potential lamin A/C phosphorylation sites in conjunction with in vitro phosphorylation assays, we determined the lamin A/C phosphoepitope(s) recognized by P-STM. We found that phosphorylation of Thr-19 is required for generating the P-STM phosphoepitope in lamin A/C and showed that it could be created in vitro by p34cdc2/cyclin B kinase (CDK1)-catalyzed phosphorylation of lamin A/C immunoprecipitated from unsynchronized HeLa S3 cells. To further explore changes in lamin A/C phosphorylation in living cells, we precisely quantified the phosphorylation levels of Thr-19 and other sites in lamin A/C isolated from HeLa S3 cells at interphase and mitosis using the SILAC method and liquid chromatography-tandem mass spectrometry. The results showed that the levels of phosphorylated Thr-19, Ser-22 and Ser-392 in both lamins A and C, and Ser-636 in lamin A only, increased -2- to 6-fold in mitotic HeLa S3 cells.
  
  
  CONCLUSIONS
  Collectively, our results demonstrate that P-STM is a useful tool for detecting Thr-19-phosphorylated lamin A/C in cells and reveal quantitative changes in the phosphorylation status of major lamin A/C phosphorylation sites during mitosis.
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
authors.forename: Jeng-Ting
authors.initials: JT
authors.lastname: Chen
authors.forename: Chia-Wen
authors.initials: CW
authors.lastname: Ho
authors.forename: Lang-Ming
authors.initials: LM
authors.lastname: Chi
authors.forename: Kun-Yi
authors.initials: KY
authors.lastname: Chien
authors.forename: Ya-Ju
authors.initials: YJ
authors.lastname: Hsieh
authors.forename: Shih-Jie
authors.initials: SJ
authors.lastname: Lin
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D034904:Lamin Type A / Q000737:chemistry / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry
chemical_list: D000906:Antibodies; D002247:Carbon Isotopes; D034904:Lamin Type A; D010748:Phosphopeptides; C440308:lamin C; D016203:CDC2 Protein Kinase