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...
A number of cellular proteins have been identified as caspase targets during cell death, including the PITSLRE protein kinases. These targets generally fall into one of three possible categories: 1) other caspases, 2) proteins that are inactivated during apoptosis, and 3) proteins that are required for execution of the cell death program. However, not all proteins are cleaved by caspases during apoptosis. Why only specific proteins are destined to be processed by caspases during cell death is currently not clear. Here we show that multiple caspase-like activities are involved in the processing of the PITSLRE p110 isoforms during Fas-induced apoptosis in Jurkat T-cells. Three p110 caspase cleavage sites have been mapped to the amino-terminal domain of p110 and verified by site-directed mutagenesis. Curiously, the mutagenesis studies revealed that cleavage of two juxtaposed caspase sites is necessary for the complete processing of this protein during cell death in vivo. Finally, we demonstrate that the PITSLRE p110 protein is rapidly phosphorylated during Fas-induced apoptosis in Jurkat cells and that phosphorylation of an aminoterminal portion of the protein may enhance caspase cleavage in this region.
Cdc2L1 and Cdc2L2 span ∼140 kb on human chromosome 1p36.3. The products of the Cdc2L genes encode almost identical protein kinases, the PITSLRE kinases, which have functions that may be relevant to the regulation of transcription/splicing and apoptotic signaling. These genes are deleted/translocated in neuroblastomas with MYCN gene amplification, a subset of malignant melanomas, and in a newly delineated deletion syndrome. Here we report that the p36.3 region of human chromosome 1 consists of two identical genomic regions, each of which contain a Cdc2L gene linked to a metalloprotease (MMP) gene in a tail-to-tail configuration. This duplicated genomic region is also linked tightly to D1Z2, a genetic marker containing a highly polymorphic VNTR (variable number tandem repeat) consisting of an unusual 40-bp reiterated sequence. Thus, these genes and the polymorphic marker D1Z2 are organized as follows: telomere-D1Z2-5Ј-MMP22-3Ј-3Ј-Cdc2L2-5Ј-5Ј-Cdc2L1-3Ј-3Ј-MMP21-5Ј-centromere. Remarkably, the introns and exons of Cdc2L1 and Cdc2L2, as well as their flanking regions, are essentially identical. A total of 15 amino acid differences, 12 nonconservative and 3 conservative, can be found in the 773-786 amino acids specified by the various products of the Cdc2L genes. Two separate promoter/5Ј untranslated (UT) regions, CpG1 and CpG2, are identical to a reported previously methylated genomic CpG sequence and are used to express >20 different Cdc2L transcripts from the two genes. The expression of CpG2 transcripts from Cdc2L1 and Cdc2L2 is tissue/cell-line specific. CpG1 transcripts are expressed ubiquitously from both genes, with perhaps some bias towards the expression of CpG1 Cdc2L1 mRNAs in certain hematopoietic cells.[The sequence data described in this paper have been submitted to the GenBank data library under the following accession nos.: (cDNAs) AF067511-AF067529; (genomic) AF080678-AF080697.]The human Cdc2L1 and Cdc2L2 genes were localized previously to chromosome band 1p36.3, and they are deleted/altered frequently in neuroblastomas with amplified MYCN genes, a subset of malignant melanoma, and a 1p36 deletion disorder Shapira et al. 1997;Nelson et al. 1998). In fact, these genes are in close proximity to p73, which like Cdc2L1/2 is located proximally to D1Z2 (Kaghad et al. 1997). The Cdc2L1/2 genes express >20 distinct PITSLRE protein kinase isoforms, many arising by alternative splicing ; this study). Indirect immunofluorescence has demonstrated that the larger p110 PITSLRE isoforms expressed by Cdc2L1 and Cdc2L2 are localized in the nucleus, with a high concentration in nuclear speckles (Loyer et al. 1998). The cell cycle-regulated RNA-binding protein RNPS1 was isolated with the PITSLRE p110 isoforms by two-hybrid cloning (Loyer et al. 1998). Overexpression of RNPS1 in mammalian cells results in the redistribution of this kinase, RNPS1, and certain splicing components into fewer, and much larger, nuclear speckles. Treatment of the same mammalian cells with transcriptional inhibitors (e.g., H8, DRB) results in a...
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