Identification of a dominant negative functional domain on DAPK-1 that degrades DAPK-1 protein and stimulates TNFR-1-mediated apoptosis.
DAPK-1 is a stress-activated tumor suppressor protein that plays a role in both proapoptotic or antiapoptotic signal transduction pathways. To define mechanisms of DAPK-1 protein regulation, we have determined that DAPK-1 protein has a long half-life, and therefore its activity is primarily regulated at the protein level. Changes in DAPK-1 protein levels occur by a cathepsin B-dependent pathway, prompting us to evaluate whether cathepsin B plays positive or negative role in DAPK-1 function. The transfection of p55-TNFR-1 induced complex formation between DAPK-1 and cathepsin B. Depletion of cathepsin B protein using small interfering RNA stimulated TNFR-1 dependent apoptosis. The minimal binding region on DAPK-1 for cathepsin B was mapped to amino acids 836-947. The transfection of the DAPK-1-(836-947) miniprotein acted in a dominant negative manner inducing endogenous DAPK-1 protein degradation in a TNFR-1-dependent manner. These data suggest that DAPK-1 forms a multiprotein survival complex with cathepsin B countering the rate of TNFR-1-dependent apoptosis and highlights the importance of developing DAPK-1 inhibitors as agents to sensitize cells to stress-induced apoptosis.
Lin
Yao
Y
University of Edinburgh, CRUK p53 Signal Transduction Group, South Crewe Road, Edinburgh EH4 2XR, Scotland, United Kingdom.
Stevens
Craig
C
Hupp
Ted
T
D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D017931:DNA Primers; D064550:Death-Associated Protein Kinases; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D034741:RNA, Small Interfering / Q000502:physiology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; D017931:DNA Primers; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases