Work

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

Year: 2008

Type: article

Abstract: 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.

Source: Journal of Biological Chemistry

DOI: 10.1074/jbc.m706040200