IDENTIFICATION OF NOVEL PHOSPHORYLATION SITES IN MURINE A-TYPE LAMINS

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and micro-sequencing of peptide s spanning the almost complete lamin sequence. We show that two distin ct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and pro tein kinase C (PKC), phosphorylate murine A-type lamins at the non-alp ha-helical amino- and carboxy-terminal domains in vitro and in vivo. C dc2 kinase, but not PKC, is capable of inducing depolymerization of th e nuclear lamina in permeabilized cells. Accordingly, lamins were prop osed to be direct in vivo substrates of cdc2 kinase and PKC with diffe rent effects on the lamina dynamics. Analysis of the original A-type l amins revealed phosphorylation of residues Ser5 and Ser392. Residue Se r392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-spec ific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vi vo, a mitosis-specific phosphorylation at the cdc2-kinase-specific pho sphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC con sensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-depen dent kinase activities act as lamin kinases in mitosis and in interpha se. Other kinases may cooperate with cdc2 kinase during reversible dis assembly in mitosis and may modulate the supramolecular assembly of la min filaments.