Craig Stevens

Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src–LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.

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.

Death‐associated protein kinase (DAPK) and signal transduction: additional roles beyond cell death

Death‐associated protein kinase (DAPK) is a stress‐regulated protein kinase that mediates a range of processes, including signal‐induced cell death and autophagy. Although the kinase domain of DAPK has a range of substrates that mediate its signalling, the additional protein interaction domains of DAPK are relatively ill defined. This review will summarize our current knowledge of the DAPK interactome, the use of peptide aptamers to define novel protein–protein interaction motifs, and how these new protein–protein interactions give insight into DAPK functions in diverse cellular processes, including growth factor signalling, the regulation of autophagy, and its emerging role in the regulation of immune responses.

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

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 RNA-mediated 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) and signal transduction: blebbing in programmed cell death

Death‐associated protein kinase (DAPK) regulates many distinct signalling events, including apoptosis, autophagy and membrane blebbing. The role of DAPK in the blebbing process is only beginning to be understood and, in this review, we will first summarize what is known about the cytoskeletal proteins and signalling cascades that participate in bleb growth and retraction and then highlight how DAPK integrates with these processes. Membrane blebs are quasispherical cellular protrusions that have a lifetime of approximately 2 min. During expansion, blebs are initially devoid of actin, although actomyosin contractions provide the motive force for growth. Once growth slows, an actin cortex reforms and actin‐bundling and contractile proteins are recruited. Finally, myosin contraction powers bleb retraction into the cell body. Blebbing occurs in a variety of cell types, from cancerous cells to embryonic cells, and can be seen in cellular phenomena as diverse as cell spreading, movement, cytokinesis and cell death. Although the machinery that executes this is still undefined in detail, the conservation of blebbing phenomenon suggests a fundamental role in metazoans and DAPK offers a door to further dissect this fascinating process.

The intermediate filament protein, vimentin, is a regulator of NOD2 activity

Objective Mutations in the nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) gene remain the strongest genetic determinants for Crohns disease (CD). Having previously identified vimentin as a novel NOD2-interacting protein, the authors aimed to investigate the regulatory effects of vimentin on NOD2 function and the association of variants in Vim with CD susceptibility. Design Coimmunoprecipitation, fluorescent microscopy and fractionation were used to confirm the interaction between NOD2 and vimentin. HEK293 cells stably expressing wild-type NOD2 or a NOD2 frameshift variant (L1007fs) and SW480 colonic epithelial cells were used alongside the vimentin inhibitor, withaferin A (WFA), to assess effects on NOD2 function using the nuclear factor-kappaB (NF-κB) reporter gene, green fluorescent protein-LC3-based autophagy, and bacterial gentamicin protection assays. International genome-wide association meta-analysis data were used to test for associations of single-nucleotide polymorphisms in Vim with CD susceptibility. Results The leucine-rich repeat domain of NOD2 contained the elements required for vimentin binding; CD-associated polymorphisms disrupted this interaction. NOD2 and vimentin colocalised at the cell plasma membrane, and cytosolic mislocalisation of the L1007fs and R702W variants correlated with an inability to interact with vimentin. Use of WFA demonstrated that vimentin was required for NOD2-dependent NF-κB activation and muramyl dipeptide-induced autophagy induction, and that NOD2 and vimentin regulated the invasion and survival properties of a CD-associated adherent-invasive Escherichia coli strain. Genetic analysis revealed an association signal across the haplotype block containing Vim. Conclusion Vimentin is an important regulator of NOD2 function and a potential novel therapeutic target in the treatment of CD. In addition, Vim is a candidate susceptibility gene for CD, supporting the functional data.

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.

A Germ Line Mutation in the Death Domain of DAPK-1 Inactivates ERK-induced Apoptosis

p53 is activated genetically by a set of kinases that are components of the calcium calmodulin kinase superfamily, including CHK2, AMP kinase, and DAPK-1. In dissecting the mechanism of DAPK-1 control, a novel mutation (N1347S) was identified in the death domain of DAPK-1. The N1347S mutation prevented the death domain module binding stably to ERK in vitro and in vivo. Gel filtration demonstrated that the N1347S mutation disrupted the higher order oligomeric nature of the purified recombinant death domain miniprotein. Accordingly, the N1347S death domain module is defective in vivo in the formation of high molecular weight oligomeric intermediates after cross-linking with ethylene glycol bis(succinimidylsuccinate). Full-length DAPK-1 protein harboring a N1347S mutation in the death domain was also defective in binding to ERK in cells and was defective in formation of an ethylene glycol bis(succinimidylsuccinate)-cross-linked intermediate in vivo. Full-length DAPK-1 encoding the N1347S mutation was attenuated in tumor necrosis factor receptor-induced apoptosis. However, the N1347S mutation strikingly prevented ERK:DAPK-1-dependent apoptosis as defined by poly(ADP-ribose) polymerase cleavage, Annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling imaging. Significant penetrance of the N1347S allele was identified in normal genomic DNA indicating the mutation is germ line, not tumor derived. The frequency observed in genomic DNA was from 37 to 45% for homozygous wild-type, 41 to 47% for heterozygotes, and 12 to 15% for homozygous mutant. These data highlight a naturally occurring DAPK-1 mutation that alters the oligomeric structure of the death domain, de-stabilizes DAPK-1 binding to ERK, and prevents ERK:DAPK-1-dependent apoptosis.

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

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.

Inflammatory bowel disease drugs: A focus on autophagy

Inflammatory bowel disease [IBD] is characterized by chronic inflammation of the gastrointestinal tract. Medications such as corticosteroids, thiopurines, immunomodulators and biologic agents are used to induce and maintain remission; however, response to these drugs is variable and can diminish over time. Defective autophagy has been strongly linked to IBD pathogenesis, with evidence showing that enhancing autophagy may be therapeutically beneficial by regulating inflammation and clearing intestinal pathogens. It is plausible that the therapeutic effects of some IBD drugs are mediated in part through modulation of the autophagy pathway, with studies investigating a wide range of diseases and cell types demonstrating autophagy pathway regulation by these agents. This review will highlight the current evidence, both in vitro and in vivo, for the modulation of autophagy by drugs routinely used in IBD. A clearer understanding of their mechanisms of action will be invaluable to utilize these drugs in a more targeted and personalized manner in this diverse and often complex group of patients.