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 phospho rylated 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-kina se 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 concom itant 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 phosph orylation of the motif in vivo positively regulates nuclear localizati on together with the nuclear localization sequence. (iii) The assembly of lamin A into the perinuclear lamina is disturbed by mutation of th e carboxy-terminal Ser-525, previously shown to be interphase-specific ally phosphorylated (Eggert et al., Eur. J. Biochem. 213, 659-671 (199 3)). The phenotype shows discontinuous and patch-like aggregates of th e mutant protein in the nucleus. We suggest that phosphorylation of th e site either regulates lamina assembly or lamina-chromatin interactio n in interphase.