DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing.
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.
Harrison
Ben
B
University of Edinburgh Cancer Centre, Cell Signalling Unit, South Crewe Road, Edinburgh, Scotland, United Kingdom.
Kraus
Michaela
M
Burch
Lindsay
L
Stevens
Craig
C
Craig
Ashley
A
Gordon-Weeks
Phillip
P
Hupp
Ted R
TR
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D020816:Amino Acid Motifs / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D064550:Death-Associated Protein Kinases; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
D000199:Actins; D051017:Apoptosis Regulatory Proteins; C475491:MAP1B protein, human; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D019151:Peptide Library; D010455:Peptides; D014404:Tubulin; C025946:3-methyladenine; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000225:Adenine