DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing
Authors: Ted R Hupp
Ben Harrison
Lindsay Burch
Phillip Gordon-Weeks
Michaela Kraus
Craig Stevens
Ashley Craig
Journal: Journal of Biological Chemistry (2008)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: DAPK-1; protein-protein interactions; peptide libraries; MAP1B; immunobinding assays
Affiliations: University of Edinburgh Cancer Centre, Cell Signalling Unit, South Crewe Road, Edinburgh, Scotland, United Kingdom.

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. 

Retracted: No

PubPeer comments:
 Figure 1b. Much more similar after horizontal flip than expected.  , 
 The authors agree that this data look very similar and the data could have been inadvertently flipped when compiling the figure. The original blots are no longer available for orientation purposes. However, if the image was flipped, this would not change the outcome of this figure. The aim of this figure was to show that purified DAPK was active (Fig 1A, E1 and E2 fractions). The E1 and E2 fractions were both shown to be active as protein kinases (Fig 1B top panel, phospho-p53 bands, lanes 4, 5, 9, and 10). The total input protein (DO-1 reactive protein) is in the bottom panel of Fig 1B and this is the data in question. This is an internally controlled experiment where the same amount of DO-1 reactive p53 substrate was added to each of the kinase reactions in lanes 1-10. So that if the image was inadvertently flipped, it would not change the outcome of the experiment because p53 should be equivalent in all 10 lanes in the bottom panel of Fig 1b; the outcome being, in Figure 1B, top panel, that lanes 4 and 5 (E1 fraction of DAPK) and lanes 9 and 10 (E2 fraction of DAPK) exhibit the conditions that reveal the highest kinase activity. This active kinase was then used for further experiments in Figure 2 (phage peptide display screen). ,