Identification of a Dominant Negative Functional Domain on DAPK-1 That Degrades DAPK-1 Protein and Stimulates TNFR-1-mediated Apoptosis

Yao Lin,Craig Stevens,Ted Hupp

doi:10.1074/jbc.m611559200

Abstract

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.

Highlights

There are over 12 phosphorylation sites on p53 that alter transactivation, nuclear shuttling, and degradation, only two known biochemical activities are directly affected: sequence-specific DNA binding and p300 co-activator stability
In general the protein levels of DAPK-1 are consistent with the mRNA levels, HeLa cells possesses more DAPK-1 protein compared with A549 cells, whereas A549 cells have the highest mRNA level (Fig. 1A)
This indicates that DAPK-1 can be regulated post-translationally, and this regulation may be important for determining the basal protein levels of DAPK-1

Summary

Introduction

There are over 12 phosphorylation sites on p53 that alter transactivation, nuclear shuttling, and degradation, only two known biochemical activities are directly affected: sequence-specific DNA binding and p300 co-activator stability. The depletion of cathepsin B protein using siRNAstimulated TNFR-1-dependent apoptosis suggesting that this DAPK-1-containing multiprotein complex can regulate negatively the rate of TNFR-1-dependent cell death pathways. The half-life of DAPK-1 protein was subsequently examined cathepsin B inhibitor with MG132 on inhibition of the cleavage in HCT116 p53ϩ/ϩ cells by performing a time course of cyclo- of DAPK-1 (Fig. 2E).

Results

Conclusion