Identification of protein kinase C (PKC) phosphorylation sites on human lamin B: Potential role of PKC in nuclear lamina structural dynamics

Barbara A. Hocevar, David J. Burns, Alan P. Fields

Research output: Contribution to journalArticlepeer-review

116 Scopus citations


Protein kinase C (PKC) is activated at the nuclear membrane in response to a variety of mitogenic stimuli. In human leukemic cells, the βII 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 βII PKC and isolated nuclear envelopes from the human promyelocytic (HL60) leukemia cell line, we have now determined the major sites for βII 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, βII PKC-mediated phosphorylation of these sites leads to the time-dependent solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro. βII 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 βII PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina structure in vitro. Nuclear βII PKC, like p34cdc2 kinase, may function to regulate nuclear lamina structural stability during cell cycle.

Original languageEnglish (US)
Pages (from-to)7545-7552
Number of pages8
JournalJournal of Biological Chemistry
Issue number10
StatePublished - Apr 5 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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