Protein kinase C (PKC) is activated at the nuclear membrane in response to a variety of mitogenic stimuli. In human leukemic cells, the β(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 β(II) PKC and isolated nuclear envelopes from the human promyelocytic (HL60) leukemia cell line, we have now determined the major sites for β(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 α-helical rod domain. Functionally, β(II) PKC-mediated phosphorylation of these sites leads to the time-dependent solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro. β(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 p13(suc1) Sepharose beads nor the presence of a p34(cdc2) kinase peptide substrate had any effect on PKC-mediated lamin B phosphorylation. Therefore, we conclude that β(II) PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina structure in vitro. Nuclear β(II) PKC, like p34(cdc2) kinase, may function to regulate nuclear lamina structural stability during cell cycle.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology