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Biochem. J. (2001) 358 (705–715) (Printed in Great Britain)

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

Anja KRIPPNER-HEIDENREICH*¹, Robert V. TALANIAN†, Renate SEKUL‡, Regine KRAFT§, Hubert THOLE², Holger OTTLEBEN‡ and Bernhard LÜSCHER¶³

*Institut für Molekularbiologie, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, 30623 Hannover, Germany, †Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605-4314, U.S.A., ‡Graffinity GmbH, Im Neuenheimer Feld 517-518, 61920 Heidelberg, Germany, §Max-Delbrück-Centrum, Proteinchemie, 13092 Berlin, Germany, Kinderklinik, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, 30623 Hannover, Germany, and ¶Abteilung Biochemie und Molekularbiologie, Institut für Biochemie, Klinikum, RWTH, Pauwelsstrasse 30, 52057 Aachen, Germany

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 IEVE¹⁰S and one at SAFD¹³⁵G 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.

Key words: caspase-7, c-Myc, DNA binding, IC₅₀, protein kinase CK2.

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.

¹Present address: Institut für Zellbiologie und Immunologie, Universität Stuttgart, 70569 Stuttgart, Germany.

²Present address: Solvay Pharmaceuticals GmbH, 30002 Hannover, Germany.

³To whom correspondence should be addressed at Abteilung Biochemie und Molekularbiologie (e-mail luescher@rwth-aachen.de).

Received 16 May 2001/27 June 2001; accepted 16 July 2001

The Biochemical Society, London © 2001