Regulation of caspase-6 and FLIP by the AMPK family member ARK5

Abstract

Colorectal cancer cells are unique in that they escape Fasmediated cell death in the presence of Fas ligand, and we recently reported that AMP-activated protein kinaserelated kinase 5 (ARK5) suppresses cell death signaling mediated by cell death receptor in Akt-dependent manner. In the current study, therefore, we examined whether ARK5 is involved in the escape from Fas-mediated cell death of colorectal cancer cells. Among 10 cell lines, ARK5 mRNA expression was observed in LoVo, SW480, and SW1116 cell lines. Interestingly, SW480 and SW1116 cell lines, but not LoVo cell line, showed expressions of both Fas ligand (FasL) and Fas mRNAs. SW620 cell line also showed FasL mRNA; however, Fas and ARK5 mRNAs were not detected. Furthermore, wellcoincided expression among ARK5, FasL, and Fas mRNAs was observed in tumor tissues from patients with colorectal cancer, suggesting the suppression of FasL/Fas system-induced cell death by ARK5 in colorectal cancer cell lines. Intensive cell death, which was dependent on the FasL/Fas system was encountered when ARK5 antisense RNA (ARK5/AS) was introduced into SW480 cells. FLIP was expressed in only ARK5 mRNA-expressing cell lines, and ARK5/AS induced FLIP cleavage in a caspase-6-dependent manner. Amino-acid sequence analysis of caspase-6 revealed two putative sites of phosphorylation by ARK5 at Ser 80 and Ser 257 . Although active caspase-6 overexpression induced cell death in SW480 and DLD-1 cell lines, SW480 cells, but not DLD-1 cells, exhibited strong resistance to procaspase-6 overexpression. Moreover, mutant caspase-6, in which the Ser 257 was substituted by Ala (caspase-6/SA), induced cell death and FLIP degradation, even in SW480 cells. Active ARK5 was found to phosphorylate wild-type caspase-6 in vitro, but not caspase-6/SA, and the prevented activation of caspase-6 was promoted due to its phosphorylation by active ARK5 in vitro. On the basis of the results of this study, we propose that ARK5 negatively regulates procaspase-6 by phosphorylation at Ser 257 , leading to resistance to the FasL/Fas system.

Cellular Mechanisms Controlling Caspase Activation and Function

“…Caspase-6 can be inhibited posttranslationally by kinase ARK5 (Suzuki et al 2004). In the colon cancer cell line, SW480, which effectively evades Fas-induced apoptosis, it has been shown that caspase-6 is held inactive by the ARK5 kinase.…”

“…Although there are two putative ARK5 sites on capase-6, the kinase appears to phosphorylate Ser257. Mutation of Ser257 to Ala overrides ARK5-mediated inhibition and allows c-FLIP cleavage and apoptosis to proceed in the SW480 cells (Suzuki et al 2004). …”

Zinc-mediated Allosteric Inhibition of Caspase-6

“…Caspase-6 differs in several respects from the other executioner caspases. Caspase-6 is much more weakly apoptotic than caspase-3 and -7, although overexpression of caspase-6 does result in apoptosis (2). Caspase-6 also cleaves a different set of cellular substrates (3,4) and displays different substrate specificity against peptide substrates (5).…”

Palmitoylation of caspase-6 by HIP14 regulates its activation

“…5,27,28 CASP6 activity can furthermore be regulated at both transcriptional and posttranslational levels, including transcriptional initiation, alternate splicing, and phosphorylation events. [29][30][31][32][33] However, it remains unclear which mechanisms are responsible for the increased CASP6 activation in neurodegenerative diseases, such as HD.…”