Abstract
BackgroundLamins 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.ResultsHere, 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.ConclusionsCollectively, 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.
Highlights
Lamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins
Phosphorylation of interphase lamin A/C by CDK1 Creates a P-STM phosphoepitope We have previously shown that lamins A and C are two of the mitotic phosphoproteins recognized by the P-STM antibody [21]
Because the CDK1 complex is known to phosphorylate lamin A/C at multiple sites during mitosis [3,4,5,6], we tested whether interphase lamin A/C can be phosphorylated in vitro by CDK1 to create a P-STM phosphoepitope
Summary
Lamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. The nuclear lamina connects with both integral membrane proteins of the inner nuclear membrane and chromatin [1]. Nuclear lamins are intermediate filament proteins that are the major components of the nuclear lamina. Lamins are type V intermediate filament proteins consisting of a central coiled-coil region [3,4,5,6,7,8,9]. Multiple cell cycle-dependent lamin C phosphorylation sites have been determined [5]. Mutation of two lamin A phosphorylation sites, Ser-22 (in QASSTP LS22PTRIT) and Ser-392 (in RLRLSPS392PTSQR), has been reported to prevent nuclear lamina disassembly in mitotic cells [3]
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