Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1

Max is the central component of the Myc/Max/Mad network of transcription fa ctors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitu tive and no posttranslational regulation is known. We have found that Max i s 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 IEVE(10)down arrowS and one at SAFD(135)down arrowG near the C-terminus, which are cleaved in vitro by caspase-5 and ca spase-7 respectively. Mutational analysis indicates that both sites are als o used in vivo. Thus Max represents the first caspase-5 substrate. The unus ual cleavage after a glutamic acid residue is observed only with full-lengt h, DNA-binding competent Max protein but not with corresponding peptides, s uggesting 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 phosphoryla tion site in vivo. These findings suggest that Fas-mediated dephosphorylati on of Max is required for cleavage by caspase-5. The modifications that occ ur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct pro cesses, namely dephosphorylation and cleavage by caspase-5 and caspase-7, t hat target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.