Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE10↓S and one at SAFD135↓G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.
- DNA binding
- protein kinase CK2
↵1 Present address: Institut für Zellbiologie und Immunologie, Universität Stuttgart, 70569 Stuttgart, Germany.
↵2 Present address: Solvay Pharmaceuticals GmbH, 30002 Hannover, Germany.
Abbreviations used: EMSA, electrophoretic mobility-shift assay; bHLHZip, basic region/helix-loop-helix/leucine zipper; ODC, ornithine decarboxylase; PARP, poly(ADP-ribose) polymerase; USF, upstream stimulatory factor.
- The Biochemical Society, London ©2001