Cleavage of p21Cip1/Waf1 and p27Kip1 Mediates Apoptosis in Endothelial Cells through Activation of Cdk2: Role of a Caspase Cascade

Abstract

Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic up-regulation of cyclin A-associated cyclin-dependent kinase 2(cdk2) activity. In apoptotic cells, the C termini of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 are truncated by specific cleavage. The enzyme involved in this cleavage is CPP32 and/or a CPP32-like caspase. After cleavage, p21Cip1/Waf1 loses its nuclear localization sequence and exits the nucleus. Cleavage of p21Cip1/Waf1 and p27Kip1 results in a substantial reduction in their association with nuclear cyclin-cdk2 complexes, leading to a dramatic induction of cdk2 activity. Dominant-negative cdk2, as well as a mutant of p21Cip1/Waf1 resistant to caspase cleavage, partially suppress apoptosis. These data suggest that cdk2 activation, through caspase-mediated cleavage of cdk inhibitors, may be instrumental in the execution of apoptosis following caspase activation.

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

“…This observation is in total agreement with previous reports showing that the amino terminus of p27 is su cient to bind and to inhibit the cyclin/CDK2 complexes (Luo et al, 1995). In contrast, our data di er from Levkau's study (Levkau et al, 1998) showing that the cleavage of p21 and p27 by a CPP32-like caspase leads to their dissociation from cyclin/CDK2 complexes and to the activation of cyclin A/CDK2. Furthermore, we found that p23 was ®ve to ten times more e cient than p27 in inhibiting CDK2 activity.…”

“…This hypothesis is supported by the fact that a putative caspase-3 consensus cleavage sequence exists within p27 sequence (Asp 136 -Ser-Ser-Asp 139 in murine p27 and Asp 136 -Pro-Ser-Asp 139 in human p27). This site can be cleaved in vitro by CCP32 and generates a fragment of 22 ± 23 kDa (Levkau et al, 1998). The analysis of the p27 polypeptidic sequence reveals that this cleavage results in the removal of the nuclear localization signal (Lys 153 to Arg 169 ) and of the CDK2 phosphorylation consensus site Thr 187 -Pro-Lys-Lys 190 at position 187 in the carboxyl terminus.…”

“…However these cells express constitutive levels of p27 and therefore do not allow the approach of speci®c biological function of p23. Based upon the results obtained with the caspase inhibitors ( Figure 5), and given the presence of a 136 DXXD 139 consensus caspase site in p27 sequence (Levkau et al, 1998;Polyak et al, 1994b;Talanian et al, 1997), we have constructed a mammalian expression vector containing the cDNA sequence of the ®rst 408 base pairs of human p27, coding for the 23 kDa fragment comprised between Met 1 and Asp 139 (Figure 7 panel a). This construction was used to express p23 into the osteosarcoma cell line SaOS-2 that does not exhibit endogenously p27 (Toyoshima and Hunter, 1994).…”

Cyclin E induction by genotoxic stress leads to apoptosis of hematopoietic cells

“…Induction of apoptosis by various stimuli has been shown to require activation of Cdk2 (Levkau et al, 1997;Meikrantz & Schlegel, 1996), while expression of Cdk inhibitors in neurons (Park et al, 1998), or during myocyte di erentiation (Wang and Walsh, 1996), prevented apoptosis. However, in contrast to our studies, cyclin A and not cyclin E-dependent kinases have been implicated in apoptosis of these cells.…”

Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis

“…Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli (Matthess et al, 2010). Cdk2 activation is mainly detected in apoptotic cells that exhibit G 1 /S phase arrest, as observed in ultraviolet irradiation-treated mesangial cells (Hiromura et al, 2002), etoposide-induced human leukemic cells (Choi et al, 2007;Bastin-Coyette et al, 2011), growth factor-deprived HUVEC cells (Levkau et al, 1998), G-Rh2-induced human hepatoma cells (Jin et al, 2000), panaxadiol-induced SK-HEP-1 cells (Jin et al, 2003). In previous reports, we showed that Cdk2 but not Cdc2 is selectively up-regulated and this upregulation is required for the induction of apoptosis in several cancer cell lines, including SK-HEP-1 cells and HeLa cells induced by treatment with ginsenoside-Rh2 (G-Rh2) (Jin et al, 2000) or panaxadiol (Jin et al, 2003).…”