IDENTIFICATION OF NUCLEAR BETA(II) PROTEIN-KINASE-C AS A MITOTIC LAMIN KINASE

Multisite phosphorylation of the nuclear lamins is thought to regulate the process of mitotic nuclear envelope breakdown in vivo. Here we in vestigate the involvement of two proposed human mitotic lamin kinases, beta(II) protein kinase C (PKC) and p34(cdc2)/cyclin B kinase, in hum an lamin B-1 phosphorylation in vitro and in intact cells. We find tha t both kinases can phosphorylate purified soluble lamin B at similar r ates. However, beta(II) PKC phosphorylates interphase nuclear envelope lamin B at more than 200 times the rate of human p34(cdc2)/cyclin B k inase. beta(II) PRC-mediated phosphorylation of lamin B is confined to two sites, Ser(395) and Ser(405), within the carboxyl-terminal domain , whereas human p34(cdc2)/cyclin B kinase phosphorylates a single site , Ser(23), in the amino-terminal domain. A second potential p34(cdc2)/ cyclin B kinase site within the carboxyl-terminal domain, Ser(393), is not phosphorylated by human p34(cdc2)/cyclin B kinase. However, inver tebrate p34(cdc2)/cyclin B kinase from sea star exhibits a different s pecificity, phosphorylating both amino- and carboxyl-terminal sites. M itotic human lamin B from intact cells is phosphorylated predominantly in its carboxyl-terminal domain. Comparative tryptic phosphopeptide m apping demonstrates that the beta(II) PKC site, Ser(405), is a promine nt target of mitotic lamin B phosphorylation in vivo. beta(II) PKC tra nslocates to the nucleus during the G(2)/M phase of cell cycle concomi tant with phosphorylation of Ser(405), indicating a physiologic role f or nuclear beta(II) PKC activation in mitotic lamin B phosphorylation in vivo. The presence of phosphorylation sites within the carboxyl-ter minal domain of mitotic lamin B which are not phosphorylated by either beta(II) PKC or p34(cdc2)/cyclin B kinase suggests the involvement of other lamin kinase(s) in G(2)/M phase lamin B phosphorylation.