Functional analysis of phosphorylation sites in human lamin A controlling lamin disassembly, nuclear transport and assembly.
We have constructed point mutations in human lamin A cDNA at conserved serine and threonine residues, some of which were shown to be phosphorylated in vitro by cdc2-kinase and protein kinase C and in vivo. Using a functional in vivo assay system, we identified three categories of mutant phenotypes. (i) Dominant negative phenotypes in mitosis result from mutation of Thr-19 and Ser-22 within the amino-terminal cdc2-kinase motif of lamin A. An increase of aberrant mitotic phenotypes in the double mutants Thr-19/Ser-392 and Ser-22/Ser-392 suggests that concomitant phosphorylation of the three residues regulates mitotic lamin A disassembly. (ii) Mutation of both Ser-403/Ser-404 within a PKC motif flanking the nuclear localization signal inhibits transport of mutant lamin A to the nucleus in 64% of the cells. It is proposed that phosphorylation of the motif in vivo positively regulates nuclear localization together with the nuclear localization sequence. (iii) The assembly of lamin A into the perinuclear lamina is disturbed by mutation of the carboxy-terminal Ser-525, previously shown to be interphase-specifically phosphorylated (Eggert et al., Eur. J. Biochem. 213, 659-671 (1993)). The phenotype shows discontinuous and patch-like aggregates of the mutant protein in the nucleus. We suggest that phosphorylation of the site either regulates lamina assembly or lamina-chromatin interaction in interphase.
Haas
M
M
Genetisches Institut, Justus-Liebig-Universität, Giessen, Germany.
Jost
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D002642:Chick Embryo; D004247:DNA / Q000032:analysis / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D034904:Lamin Type A; D034882:Lamins; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism* / Q000502:physiology*; D010641:Phenotype; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism; D012694:Serine / Q000032:analysis; D013912:Threonine / Q000032:analysis; D014162:Transfection
D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D013912:Threonine; D012694:Serine; D004247:DNA; D011493:Protein Kinase C