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J. Biol. Chem., Vol. 283, Issue 15, 9999-10014, April 11, 2008

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DAPK-1 Binding to a Linear Peptide Motif in MAP1B Stimulates Autophagy and Membrane Blebbing^*

Ben Harrison¹, Michaela Kraus, Lindsay Burch, Craig Stevens, Ashley Craig, Phillip Gordon-Weeks², and Ted R. Hupp³

From the University of Edinburgh Cancer Centre, Cell Signalling Unit, South Crewe Road, Edinburgh EH4 2XR, Scotland and The MRC Centre for Developmental Neurobiology, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, United Kingdom

DAPK-1 (death-activated protein kinase) has wide ranging functions in cell growth control; however, DAPK-1 interacting proteins that mediate these effects are not well defined. Protein-protein interactions are driven in part by linear interaction motifs, and combinatorial peptide libraries were used to identify peptide interfaces for the kinase domain of DAPK-1. Peptides bound to DAPK-1core kinase domain fragments had homology to the N-terminal domain of the microtubule-associated protein MAP1B. Immunobinding assays demonstrated that DAPK-1 can bind to the full-length human MAP1B, a smaller N-terminal miniprotein containing amino acids 1-126 and the 12-amino acid polypeptides acquired by peptide selection. Amino acid starvation of cells induced a stable immune complex between MAP1B and DAPK-1, identifying a signal that forms the endogenous complex in cells. DAPK-1 and MAP1B co-expression form a synthetic lethal interaction as they cooperate to induce growth inhibition in a clonogenic assay. In cells, two co-localizing populations of DAPK-1 and MAP1B were observed using confocal microscopy; one pool co-localized with MAP1B plus tubulin, and a second pool co-localized with MAP1B plus cortical F-actin. Reduction of MAP1B protein using short interfering RNA attenuated DAPK-1-stimulated autophagy. Transfected MAP1B can synergize with DAPK-1 to stimulate membrane blebbing, whereas reduction of MAP1B using short interfering RNA attenuates DAPK-1 membrane blebbing activity. The autophagy inhibitor 3-methyladenine inhibits the DAPK-1 plus MAP1B-mediated membrane blebbing. These data highlight the utility of peptide aptamers to identify novel binding interfaces and highlight a role for MAP1B in DAPK-1-dependent signaling in autophagy and membrane blebbing.

Received for publication, July 23, 2007 , and in revised form, January 2, 2008.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) NM_005909 [GenBank] and NM_004938 [GenBank] .

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Ph.D. studentship from the Cancer Research UK.

² Supported by the Medical Research Council.

³ Supported by a Programme Grant from the Cancer Research UK. To whom correspondence should be addressed. E-mail: ted.hupp{at}ed.ac.uk.

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				C. Stevens, Y. Lin, B. Harrison, L. Burch, R. A. Ridgway, O. Sansom, and T. Hupp Peptide Combinatorial Libraries Identify TSC2 as a Death-associated Protein Kinase (DAPK) Death Domain-binding Protein and Reveal a Stimulatory Role for DAPK in mTORC1 Signaling J. Biol. Chem., January 2, 2009; 284(1): 334 - 344. [Abstract] [Full Text] [PDF]