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

Self Cite

Death-associated protein kinase (DAPK) is a multidomain enzyme that plays a central role in autophagic and apoptotic signaling, although the protein-protein interactions regulating DAPK functions are not well defined. Peptide aptamer libraries were used to identify the tumor suppressor protein tuberin (TSC2) as a novel DAPK death domain-binding protein, and we evaluated whether DAPK is a positive or negative effector of the TSC2-regulated mammalian target of rapamycin (mTORC1) signaling pathway. Binding studies using death domain miniproteins in vitro and deletion analysis in vivo determined that the death domain of DAPK is the major site for the interaction with TSC2. Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity is stimulated by growth factor signaling. Transfection of DAPK promotes phosphorylation of TSC2 in vivo, whereas short interfering RNAmediated attenuation of DAPK reduces growth factor-stimulated phosphorylation of TSC2. DAPK-dependent phosphorylation leads to TSC1-TSC2 complex dissociation, and consequently manipulation of DAPK by transfection or short interfering RNA demonstrated that DAPK is a positive regulator of mTORC1 in response to growth factor activation. Epistatic studies suggest that DAPK functions downstream from the RAS-MEK-ERK and phosphatidylinositol 3-kinase-AKT growth factor signaling pathways. DAPK ؉/؊ mouse embryo fibroblasts have attenuated mTORC1 signaling compared with DAPK ؉/؉ counterparts, and overexpression of DAPK in DAPK ؉/؊ MEFs stimulates mTORC1 activity. These data uncover a novel interaction between DAPK and TSC2 proteins that has revealed a positive link between growth factor stimulation of DAPK and mTORC1 signaling that may ultimately affect autophagy, cell survival, or apoptosis.Death-associated protein kinase (DAPK 2 ; also known as DAPK1) is a calcium/calmodulin (CaM)-regulated serine/threonine protein kinase that plays a central role in a diverse range of signal transduction pathways, including growth factor activation, apoptosis, and autophagy. DAPK is composed of several functional domains, including a kinase domain, a CaM binding domain, eight ankyrin repeats, and a death domain (1). DAPK was originally identified as a factor that regulates apoptosis in response to various death inducing signals, including interferon-␥, TNF-␣, and transforming growth factor-␤ (1, 2). The mechanisms through which DAPK activates apoptosis include an oncogene-activated p53-dependent pathway (1), inactivation of integrin and adhesion-mediated survival signaling (3), and cytoskeletal remodeling via phosphorylation of myosin light chain 2 (4, 5). Overexpression of the death domain module by itself protects cells from death-inducing signals, demonstrating the importance of this domain for DAPK proapoptotic function (6). The death domain of DAPK regulates its proapoptotic function in part by interacting with netrin-1 receptor UNC5H2 (7), the mitogen-activated protein kinase ERK (8), and members of the tumor necrosis superfamily TNFR1 and FADD (9). Autophosphory...

Section: Discussionsupporting

confidence: 66%

“…HA-tagged DAPK-WT, ⌬CAM, and K42A vectors were a gift of Adi Kimchi (Weizmann Institute). For the generation of HA-tagged DAPK deletion mutants, stop codons were introduced, as described previously (29). FLAG-tagged TSC2 was a gift from Andrew Tee (University of Cardiff).…”

Section: Methodsmentioning

confidence: 99%

Peptide Combinatorial Libraries Identify TSC2 as a Death-associated Protein Kinase (DAPK) Death Domain-binding Protein and Reveal a Stimulatory Role for DAPK in mTORC1 Signaling

Stevens

Lin

Harrison

et al. 2009

Self Cite

“…Previous studies have shown activation of DAPK (via dephosphorylation of p-S308) leads to enhanced degradation by the proteasome (3, 10, 11) and lysosome (6). To distinguish between proteasomal and lysosomal degradation, we induced the expression of B␣ or B␦ in HEK-T-Rex cells and then treated the cells with 10 M MG132 or 100 M chloroquine for 6 h. Cells treated with proteasomal inhibitor MG132 could rescue the B␣-or B␦-induced decrease in DAPK protein levels, whereas the lysosomal inhibitor could not (Fig.…”

Section: Pp2a-b␣ and Pp2a-b␦ Holoenzymes Associate With Dapk-mentioning

confidence: 99%

Protein Phosphatase 2A (PP2A) Holoenzymes Regulate Death-associated Protein Kinase (DAPK) in Ceramide-induced Anoikis

Widau

Jin

Dixon

et al. 2010

The tumor suppressor, death-associated protein kinase (DAPK), is a Ca 2؉ /calmodulin-regulated Ser/Thr kinase with an important role in regulating cytoskeletal dynamics. Autophosphorylation within the calmodulin-binding domain at Ser-308 inhibits DAPK catalytic activity. Dephosphorylation of Ser-308 by a previously unknown phosphatase enhances kinase activity and proteasome-mediated degradation of DAPK. In these studies, we identified two holoenzyme forms of protein phosphatase 2A (PP2A), AB␣C and AB␦C, as DAPK-interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Ser-308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK levels by enhancing proteasome-mediated degradation of the kinase. Overexpression of wild type DAPK induces cell rounding and detachment in HEK293 cells; however, this effect is not observed following expression of an inactive DAPK S308E mutant. Finally, activation of DAPK by PP2A was found to be required for ceramide-induced anoikis. Together, our results provide a mechanism by which PP2A and DAPK activities control cell adhesion and anoikis.

“…However, a number of cellular studies have shown that DAPK-1 can exhibit diverse biological functions that include the following: interferon-induced cell death (16); amino acid starvation-linked autophagy and membrane blebbing (17); integrin inhibition and cell detachment (18); ERK 4 -dependent apoptosis (10,15); oncogene activation of p53 (11); induction of phosphorylation within the p53 transactivation domain (19); and genetic tumor suppressor functions (20). Furthermore, although DAPK-1 can function as a growth suppressor (21), growing evidence also indicates it can function as an anti-apoptotic factor (22)(23)(24)(25) suggesting that DAPK-1 can respond to different input signals catalyzing distinct effects on cell growth.…”

mentioning

confidence: 99%

DAPK-1 Binding to a Linear Peptide Motif in MAP1B Stimulates Autophagy and Membrane Blebbing

Harrison

Kraus

Burch

et al. 2008

Self Cite

130

106

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. Proteinprotein 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.