Cleavage of PITSLRE Kinases by ICE/CASP-1 and CPP32/CASP-3 during Apoptosis Induced by Tumor Necrosis Factor

Abstract

Emerging evidence suggests that multiple aspartatespecific cysteine proteases (caspases (CASPs)) play a crucial role in programmed cell death. Many cellular proteins have been identified as their substrates and serve as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as an effector molecule in apoptosis. PITSLRE kinases are a superfamily of Cdc2-like kinases that have been implicated in apoptotic signaling and tumorigenesis. In this paper we report that tumor necrosis factor (TNF)-mediated apoptosis is associated with a CrmA-and Bcl-2-inhibitable cleavage of PITSLRE kinases, indicating a role for CASPs. Testing of seven murine CASPs for their ability to cleave p110 PITSLRE kinase ␣2-1 in vitro revealed that only CASP-1 (ICE (interleukin-1␤-converting enzyme)) and CASP-3 (CPP32) 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 ␣2-1 occurred at Asp 393 within the sequence YVPDS, which is similar to that involved in the CASP-1-mediated cleavage of prointerleukin-1␤. TNF-induced proteolysis of PITSLRE kinases was still observed in fibroblasts from CASP-1 0/0 mice. These data implicate CASP-3 as a potentially important CASP family protease responsible for the cleavage of PITSLRE kinases during TNF-induced apoptosis.Apoptosis is a fundamental process for normal development of multicellular organisms and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis (1). Aspartate-specific cysteine proteases belonging to the interleukin-1␤-converting enzyme (ICE) 1 family, recently renamed the caspase (CASP) family (2), have been implicated as principal effectors of apoptosis, presumably by their proteolytic action on specific targets, including members of the ICE-related protease family themselves, poly(ADP-ribose) polymerase, DNA-dependent protein kinase, the 70-kDa small U1 ribonucleoprotein, lamins, protein kinase C␦, D4-GDP dissociation inhibitor, and various components of the cytoskeleton (3, 4). Many of these proteins are likely to be involved in the morphological and biochemical changes that accompany apoptosis, or in aspects of DNA damage sensing and repair, and are used as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as a downstream effector molecule in apoptosis.PITSLRE kinases are a superfamily of protein kinases related to the master mitotic protein kinase Cdc2 (4 -6). Ectopic expression of the smallest member of this superfamily has previously been shown to induce apoptosis (5). In addition, deletion of the PITSLRE gene complex and complete loss of expression of specific isoforms occur in many neuroblastoma cell lines and is frequently observed in human cancers (8 -10). Induction of apoptosis via the Fas receptor in human T cells has recently been shown to be correlated with proteol...

p73β Is Regulated by Protein Kinase Cδ Catalytic Fragment Generated in the Apoptotic Response to DNA Damage

“…Whereas most substrates of caspase-3 are inactivated, certain proteins, such as PKC␦ (19,20), PKC (24), the p21-activated kinase 2 (33), cytosolic phospholipase A2 (34), and PITSLRE kinase a2-1 (35), are activated by caspase-3-mediated proteolysis. Cleavage of PKC␦ at a DMQD/N site in the third variable region (V3) generates a 40-kDa fragment that contains the ATP-binding and kinase domains (19,20).…”

Life and death decisions: regulation of apoptosis by proteolysis of signaling molecules

“…40 Several PITSLRE isoforms including p170 and p110 are specific targets of proteolysis by caspases during apoptosis. 41 The cleavage site determined by initial mutational analysis predicts that cleavage should remove an SH2 domain and putative nuclear localization signals while preserving an intact kinase domain. 41 A more detailed analysis of p110 cleavage sites has identified two additional cleavage sites in the amino terminus and confirms that the kinase domain remains intact following cleavage.…”

“…41 The cleavage site determined by initial mutational analysis predicts that cleavage should remove an SH2 domain and putative nuclear localization signals while preserving an intact kinase domain. 41 A more detailed analysis of p110 cleavage sites has identified two additional cleavage sites in the amino terminus and confirms that the kinase domain remains intact following cleavage. 42 Curiously, PITSLRE p110 is phosphorylated on serine residue(s) in response to Fas or TNFR activation, and the phosphorylated isoform appears to be more sensitive to in vivo cleavage by caspases.…”

The C-terminal Kinase Domain of the p34cdc2-related PITSLRE Protein Kinase (p110C) Associates with p21-activated Kinase 1 and Inhibits Its Activity during Anoikis

“…3C). Binding of p110C with PAK1 in Vivo-Previous studies indicated that p110C was produced from the cleavage of some PITSLRE kinases isoforms, such as p110 PITSLRE and p58 PITSLRE (15)(16)(17)(18), and p58 PITSLRE and p110 PITSLRE were the best studied and described among the PITSLRE protein kinase family. To further determine whether p110C interacts with PAK1 in vivo and whether the interaction was specific, the HA-p110C, HA-p58 PITSLRE , or HA-p110 PITSLRE was transiently expressed in NIH3T3 cells, and pcDNA3 was also transiently expressed in NIH3T3 cells as control.…”

“…There are at least 20 PITSLRE protein kinase isoforms, which are differentially expressed in mammalian tissues and regulate diverse cellular functions, including the cell cycle control, tumorigenesis, the regulation of RNA splicing or transcription, and so on (8, 10 -11,13-26). Studies indicated that several of the PITSLRE protein kinase isoforms might serve as effectors in apoptotic signaling pathways (15)(16)(17)(18).…”