Tuberous sclerosis‐2 (TSC2) regulates the stability of death‐associated protein kinase‐1 (DAPK) through a lysosome‐dependent degradation pathway

Abstract

We previously identified a novel interaction between tuberous sclerosis-2 (TSC2) and death-associated protein kinase-1 (DAPK), the consequence being that DAPK catalyses the inactivating phosphorylation of TSC2 to stimulate mammalian target of rapamycin complex 1 (mTORC1) activity. We now report that TSC2 binding to DAPK promotes the degradation of DAPK. We show that DAPK protein levels, but not gene expression, inversely correlate with TSC2 expression. Furthermore, altering mTORC1 activity does not affect DAPK levels, excluding indirect effects of TSC2 on DAPK protein levels through changes in mTORC1 translational control. We provide evidence that the C-terminus regulates TSC2 stability and is required for TSC2 to reduce DAPK protein levels. Importantly, using a GTPase-activating protein-dead missense mutation of TSC2, we demonstrate that the effect of TSC2 on DAPK is independent of GTPase-activating protein activity. TSC2 binds to the death domain of DAPK and we show that this interaction is required for TSC2 to reduce DAPK protein levels and half-life. Finally, we show that DAPK is regulated by the lysosome pathway and that lysosome inhibition blocks TSC2-mediated degradation of DAPK. Our study therefore establishes important functions of TSC2 and the lysosomal-degradation pathway in the control of DAPK stability, which taken together with our previous findings, reveal a regulatory loop between DAPK and TSC2 whose balance can either promote: (a) TSC2 inactivation resulting in mTORC1 stimulation, or (b) DAPK degradation via TSC2 signalling under steady-state conditions. The fine balance between DAPK and TSC2 in this regulatory loop may have subtle but important effects on mTORC1 steady-state function. Abbreviations DAPK, death-associated protein kinase-1; GAP, GTPase-activating protein; IFN, interferon; 3-MA, 3-methyladenine; MEF, mouse embryonic fibroblast; mTORC1, mammalian target of rapamycin complex 1; siRNA, short interfering RNA; TNF, tumour necrosis factor; TSC2, tuberous sclerosis-2.

Sodium selenite-induced activation of DAPK promotes autophagy in human leukemia HL60 cells

“…DAPK has been reported to bind to distinct proteins, such as ERK, p38MAPK, ribosomal S6 Kinase, and myosin light chain (25,(27)(28)(29). It can simultaneously regulate apoptosis and autophagy through separate mechanisms.…”

DAPK1 as an independent prognostic marker in liver cancer

“…The activity of DAPK1 has been shown to be regulated through Src kinase (Wang et al, 2007), the Ras/MEK/ERK pathway (Anjum et al, 2005; Chen et al, 2005) and the mTORC1 pathway (Lin et al, 2011). Although extensively researched, the role of DAPK1 and its prognostic significance in liver cancer is largely unknown.…”

Quantitative and correlation analysis of the DNA methylation and expression of DAPK in breast cancer

“…The protein expression of DAPK is controlled by both ubiquitin–proteasome (Jin et al, 2002; Lee et al, 2010; Zhang, Nephew & Gallagher, 2007) and lysosome pathways (Gallagher & Blue, 2014; Lin et al, 2008, 2009, 2011; Stevens et al, 2009). It is not surprising that the mRNA and protein expression of DAPK did not correlate well (Fig.…”