Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis

Abstract

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

Figure 1

PKCδ is proteolytically activated by CPP32 in vitro. (A) PKCδ (full-length: FL), PKCδ(D327A/D330A) and PARP were labeled with [³⁵S]methionine and incubated with recombinant CPP32β. The reaction products were analyzed by SDS-PAGE and autoradiography. The kinase active PKCδ catalytic fragment (CF) and the kinase inactive PKCδCF(K-R) were labeled with [³⁵S]methionine and analyzed under similar conditions. (B) Recombinant PKCδ and PKCδ (D327A/D330A) were incubated with CPP32 and then assayed for protein kinase activity using MBP as substrate.

Figure 2

Transfection of the PKCδ catalytic fragment (CF) induces morphologic changes characteristic of apoptosis. HeLa (upper panels) and NIH3T3 (lower panels) cells were cotransfected with pSvβ-β-gal and vectors expressing: (A and C) kinase inactive PKCδCF(K-R) and (B and D) kinase active PKCδCF. Transfection was determined by X-gal staining and apoptotic cells were identified by their condensed morphology.

Figure 3

Expression of PKCδCF results in nuclear fragmentation. HeLa cells were transfected with vectors that express: (A) full-length PKCδ; (B) kinase active PKCδCF; and (C) kinase inactive PKCδCF(K-R). (D) Cells were exposed to 2 μM ara-C. The cells were fixed with paraformaldehyde and then stained with Hoechst dye. Cotransfection of PKCδFL, PKCδCF and PKCδCF(K-R) with pSvβ-β-gal demonstrated transfection efficiencies of 50, 42, and 43%, respectively. Percentage of apoptotic cells for the PKCδFL, PKCδCF, and PKCδCF(K-R) transfected populations was 2, 26, and 4%, respectively. Bar, 15 μM.

Figure 4

Overexpression of PKCδCF induces sub-G1 DNA and chromatin condensation. (A) HeLa cells were transfected with PKCδFL, PKCδCF, or PKCδCF(K-R). Cells were assessed for DNA content by flow cytometry at 48 h after transfection. The small triangle denotes G₀/G₁ DNA. (B) HeLa cells transfected with PKCδCF(K-R) (left) and PKCδCF (right) were assessed for chromatin condensation after staining with acridine orange and ethidium bromide.