Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase.
The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head-to-tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head-to-tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N- and C-terminal to the central alpha-helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head-to-tail polymers. The relative importance of N- and C-terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C-terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N-terminal site (Ser16----Ala) rendered head-to-tail polymers resistant to disassembly. These results emphasize the importance of the N-terminal end domain for lamin head-to-tail polymerization in vitro, and they demonstrate that phosphorylation-dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.
Peter
M
M
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.
Heitlinger
E
E
Häner
M
M
Aebi
U
U
Nigg
E A
EA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D034921:Lamin Type B; D034882:Lamins; D046911:Macromolecular Substances; D008938:Mitosis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D034921:Lamin Type B; D034882:Lamins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1