Protein kinase C (PKC) is activated at the nuclear membrane in response to a variety of mitogenic stimuli. In human leukemic cells, the beta II PKC isotype is selectively translocated and activated at the nucleus. We recently identified the nuclear envelope component
lamin B1 as a major substrate for nuclear PKC both in whole cells and in vitro. Using highly purified human beta II PKC and isolated nuclear envelopes from the human promyelocytic (HL60)
leukemia cell line, we have now determined the major sites for beta II PKC-mediated
lamin B phosphorylation. Using a combination of
cyanogen bromide cleavage, direct microsequencing, tryptic
phosphopeptide, and
phosphate release analyses, two major sites of PKC-mediated phosphorylation, Ser395 and Ser405, have been identified. These sites lie within the carboxyl-terminal domain of
lamin B immediately adjacent to the central alpha-helical rod domain. Functionally, beta II PKC-mediated phosphorylation of these sites leads to the time-dependent solubilization of
lamin B indicative of mitotic nuclear envelope breakdown in vitro. beta II PKC-mediated
lamin B phosphorylation is inhibited by 1) a
monoclonal antibody directed against the active site of PKC, 2) a PKC pseudosubstrate inhibitor
peptide, and 3) a PKC
peptide substrate. Two observations indicate that PKC-mediated
lamin B phosphorylation and solubilization is due to direct phosphorylation of
lamin B by PKC rather than indirect activation of a cdc2
kinase. Neither immunodepletion with p13suc1
Sepharose beads nor the presence of a p34cdc2
kinase peptide substrate had any effect on PKC-mediated
lamin B phosphorylation. Therefore, we conclude that beta II PKC represents a physiologically relevant
lamin kinase that can directly modulate nuclear lamina structure in vitro. Nuclear beta II PKC, like p34cdc2
kinase, may function to regulate nuclear lamina structural stability during cell cycle.