Protein Kinase C-βII Is an Apoptotic Lamin Kinase in Polyomavirus-transformed, Etoposide-treated pyF111 Rat Fibroblasts

Abstract

The role of protein kinase C-␤ II (PKC-␤ II ) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-␤ II specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-␤ II complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-␤ II holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-␤ I or PKC-␦, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-␤ II is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-␤ II holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-␤s inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-␤ II response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-␤ II being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-␦ being inactive or only slightly active and by PKC-␣ not combining with the lamin.A growing body of evidence suggests that the commitment to and execution of apoptosis are mediated through the phosphorylation of specific proteins by several protein kinases (1). A number of PKC 1 isoforms appear to be among these protein kinases (2, 3). Apoptogens may activate or inactivate and cause the translocation of various PKC isoforms from the cytosol onto cytoskeletal components, cytoplasmic membranes, mitochondria, and/or the nuclear envelope (4 -11); induce their migration from such subcellular structures to nucleoplasmic and/or cytosolic fractions (8, 12); or cause the PKC holoenzymes to be cleaved into N-terminal regulatory and C-terminal catalytic fragments (CFs) by several proteases, including caspases (11,(13)(14)(15). Despite suggestions that a particular PKC isoform (e.g. the novel PKC-␦ and/or its CFs) might play a pivotal role in apoptosis (13,14), the available evidence indicates the involvement and cleavage of novel PKCs (e.g. PKC-⑀ and PKC-), the classical Ca 2ϩ -stimulable PKCs (e.g. PKC-␤), atypical PKCs (e.g. PKC-), and PKC-related (e.g. PRK) kinases in apoptosis (2,3,15). This is the third report from a continuing study of the roles of protein kinase C isozymes in drug-induced apoptosis using a polyomavirus-transformed embryo rat fibroblast, the pyF111 cell, as a model (10, 11). We chose this fibroblast, since it is prone to apoptosis because it cannot make the antiapoptotic Bcl-2 and Bcl-X L proteins but can make the proapoptotic Bax protein (10, 11). We have shown that whereas a surge of the activity of PKC-␦ holopr...

Calphostin C, a remarkable multimodal photodynamic killer of neoplastic cells by selective nuclear lamin B1 destruction and apoptogenesis (Review)

“…1C) (14). Different PKCs have also been shown to be involved in apoptogenesis as well as proliferation and other activities (25,27,30,31). Therefore, as expected, when CalC ÊE is driving oxidized lamin B1 destruction, it silences PKC-ß I and PKC-‰ and any indication of impending apoptogenesis ( Fig.…”

Characterization and Intracellular Localization of the Epstein-Barr Virus Protein BFLF2: Interactions with BFRF1 and with the Nuclear Lamina

“…Of interest is that in mouse CMV, the BFRF1 homolog has been shown to recruit cellular PKC to dissolve the nuclear lamina, allowing virions to gain access to the nuclear membrane for primary envelopment (28). Since it has been shown that PKC-␤II is directly responsible for lamin B phosphorylation and solubilization (21) and that the selective PKC-␤ inhibitor hispidin reduces lamin B phosphorylation and totally prevents lamin B proteolysis (8), it might be of interest to investigate whether the use of selective PKC inhibitors might interfere with the correct intracellular localization of lamin B or with its interaction with BFRF1, ultimately leading to the lamina disassembly that is needed for the breakdown of the nuclear envelope.…”

Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

“…During apoptosis, PKC␦, a novel PKC, has been shown to translocate to the nuclear membrane and to phosphorylate lamin B at the Cterminal domain, thereby inducing apoptotic lamina disassembly (4,6,9,10). PKC␤II has also been shown to phosphorylate and thereby solubilize lamin B (8). Conversely, PKC␣ has been shown to bind lamin A protein yet functions as an apoptosis inhibitor (9,20).…”