Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C.
Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth.
Fields
A P
AP
Johns Hopkins Oncology Center, Baltimore, Maryland 21231.
Pettit
G R
GR
May
W S
WS
D054713:Bryostatins; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D034921:Lamin Type B; D034882:Lamins; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D018942:Macrolides; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*
D054713:Bryostatins; D007783:Lactones; D034921:Lamin Type B; D034882:Lamins; D018942:Macrolides; D009687:Nuclear Proteins; D010703:Phorbol Esters; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1; D011493:Protein Kinase C