Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C.

A P Fields,G R Pettit,W S May

doi:10.1016/s0021-9258(18)68471-6

Abstract

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.

Highlights

From the $Johns HopkinsOncology Center, Baltimore, Maryland 21231 and the §Cancer Research Institute and the Department of Chemistry, Arizona State University, Tempe, Arizona 85287
Entiate, bryostatin1 antagonizes thiseffect and allows as well as for diacylglycerols which are generated endogecells to continue proliferating. To test whether these divergent effectscould 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 PBtz
Dalton brane may represent a site for the differential activation of protein kinase C, we investigated the effect of both bryostatin 1and PBtz on the phosphorylation state of nuclear envelope polypeptides

Summary

RESULTS

Both PBt2 and bryostatin 1 can translocate cytosolic protein kinase C andactivate the enzyme at the plasma membrane to mediate the rapid phosphorylation and down-regulation of the cell-surface transferrin receptor [11, 12, 14, 23].previousfindingsindicate that these compounds induce phosphorylationof a similarpattern of cytosolicproteins [10]. W from cells treated for 15 min with bryostatin 1 or PBh or both by two-dimensional IEF/SDS-PAGE analysis and autoradiography reveals the presence of fouracidicnuclear envelope polypeptides whichare phosphorylated in response to bryostatin 1,either alone (Fig. 5C) or in combination with PBh (Fig. 50). No such increased phosphorylation can be seen in control (Fig. 5A) or PBb-treated (Fig. 5B) cells. Nuclear envelope polypeptides from 32P-labeled, M,of 67,000 and an acidic PI of about 6.0.A survey of the bryostatin-treated or control cells wereresolvedby two-diliterature reveals that the characteristics of lamin B The PI shift accompanying 32Plabeling of lamin B indicates that the observed phosphorylation is the result of the addition of phosphate groups to lamin B molecules, resulting in a net increase in the negative charge of the molecules rather than increased exchangeof phosphate groups already on lamin B, a process whichwould result in increased32Plabeling of lamin B without a shiftin PI

Lamin B Is a Major Substratefor Activated Protein Kinase

DISCUSSION

Activation KofinParsoetein