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 caspase-3 and not by caspase-2,-4,-5,-6 and -7. 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 d or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKCd by a caspase-3 contributes to phenotypic changes associated with apoptosis (Ghayur et al., 1996). Protein kinase C d is one of the few examples of a substrate that is cleaved by caspase-3 and not by caspase-7.

In a second paper is was shown that both ionising radiation (IR)-induced proteolytic activation of PKCd and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-x_L supporting an association with apoptosis in the treatment of human U-937 with IR. Again, cleavage of PKCd was mapped adjacent to an aspartic acid at the same QDN site. In addition the specific tetrapeptide YVAD blocked both proteolytic activation of PKCd and internucleosomal DNA fragmentation in these IR-treated cells (Emoto et al., 1995).

Additional evidence was published that proteolytic cleavage of PKC theta by caspase-3 induces events characteristic of apoptosis. Also PKC theta cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-x_L or the baculovirus p35 protein. PKC theta is cleaved by caspase-3 and by apoptotic cell lysates at a DEVD³⁵⁴K site. Overexpression of the cleaved kinase- active PKC theta fragment, but not full-length PKC theta or a kinase-inactive fragment, results in induction of sub-G1 phase DNA, nuclear fragmentation, and lethality (Datta et al., 1997).

D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of caspase-3. D4-GDI is rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by YVAD-cmk. D4-GDI was cleaved in vitro by recombinant caspase-3 expressed in Escherichia coli to form a 23-kDa fragment. The caspase-3-mediated cleavage of D4-GDI was completely inhibited by 1 m M DEVD-CHO, a reported selective inhibitor of caspase-3. In contrast, the inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit caspase-3-mediated D4-GDI cleavage at concentrations up to 50 m M. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD¹⁹S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by caspase-3-mediated cleavage of the GDI protein (Na et al., 1996)

PITSLRE kinases are a superfamily of Cdc2- like kinases that have been implicated in apoptotic signaling and tumorigenesis. Is was demonstrated that tumor necrosis factor (TNF)-mediated apoptosis is associated with a CrmA- and Bcl-2- inhibitable cleavage of PITSLRE kinases, indicating a role for caspases. Testing of seven murine caspases for their ability to cleave p110 PITSLRE kinase alpha2-1 in vitro revealed that only caspase-1 and caspase-3 were able to produce the same 43-kDa cleavage product as observed in cells undergoing TNF-induced apoptosis. Mutational analysis revealed that cleavage of p110 PITSLRE kinase alpha2-1 occurred at Asp393 within the sequence YVPDS, which is similar to that involved in the caspase-1-mediated cleavage of prointerleukin-1b . TNF-induced proteolysis of PITSLRE kinases was still observed in fibroblasts from caspase-1^0/0 mice. These data implicate caspase-3 as a potentially important caspase responsible for the cleavage of PITSLRE kinases during TNF-induced apoptosis (Beyaert et al., 1997)