GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling.

Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein.

New modularity of DAP-kinases: alternative splicing of the DRP-1 gene produces a ZIPk-like isoform.

DRP-1 and ZIPk are two members of the Death Associated Protein Ser/Thr Kinase (DAP-kinase) family, which function in different settings of cell death including autophagy. DAP kinases are very similar in their catalytic domains but differ substantially in their extra-catalytic domains. This difference is crucial for the significantly different modes of regulation and function among DAP kinases. Here we report the identification of a novel alternatively spliced kinase isoform of the DRP-1 gene, termed DRP-1ß. The alternative splicing event replaces the whole extra catalytic domain of DRP-1 with a single coding exon that is closely related to the sequence of the extra catalytic domain of ZIPk. As a consequence, DRP-1ß lacks the calmodulin regulatory domain of DRP-1, and instead contains a leucine zipper-like motif similar to the protein binding region of ZIPk. Several functional assays proved that this new isoform retained the biochemical and cellular properties that are common to DRP-1 and ZIPk, including myosin light chain phosphorylation, and activation of membrane blebbing and autophagy. In addition, DRP-1ß also acquired binding to the ATF4 transcription factor, a feature characteristic of ZIPk but not DRP-1. Thus, a splicing event of the DRP-1 produces a ZIPk like isoform. DRP-1ß is highly conserved in evolution, present in all known vertebrate DRP-1 loci. We detected the corresponding mRNA and protein in embryonic mouse brains and in human embryonic stem cells thus confirming the in vivo utilization of this isoform. The discovery of module conservation within the DAPk family members illustrates a parsimonious way to increase the functional complexity within protein families. It also provides crucial data for modeling the expansion and evolution of DAP kinase proteins within vertebrates, suggesting that DRP-1 and ZIPk most likely evolved from their ancient ancestor gene DAPk by two gene duplication events that occurred close to the emergence of vertebrates.

Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by weakening its interactions with Bcl-2 and Bcl-XL.

Beclin 1, an essential autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members. The dissociation of Beclin 1 from the Bcl-2 inhibitors is essential for its autophagic activity, and therefore is tightly controlled. We recently revealed a novel phosphorylation-based mechanism by which death-associated protein kinase (DAPk) regulates this process. We found that DAPk phosphorylates Beclin 1 on T119, a critical residue within its BH3 domain, and thus promotes Beclin 1 dissociation from Bcl-X(L) and autophagy induction. Here we report that T119 phosphorylation also reduces the interaction between Beclin 1 and Bcl-2, in line with the high degree of structural homology between the BH3 binding pockets of Bcl-2 and Bcl-X(L) proteins. Our results reveal a new phosphorylation-based mechanism that reduces the interaction of Beclin 1 with its inhibitors to activate the autophagic machinery.

DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy.

Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.

A high throughput proteomics screen identifies novel substrates of death-associated protein kinase.

Death-associated protein kinase (DAPk) is a Ser/Thr kinase whose activity is necessary for different cell death phenotypes. Although its contribution to cell death is well established, only a handful of direct substrates have been identified; these do not fully account for the multiple cellular effects of DAPk. To identify such substrates on a large scale, we developed an in vitro, unbiased, proteomics-based assay to search for novel DAPk substrates. Biochemical fractionation and mass spectrometric analysis were used to purify and identify several potential substrates from HeLa cell lysate. Here we report the identification of two such candidate substrates, the ribosomal protein L5 and MCM3, a replication licensing factor. Although L5 proved to be a weak substrate, MCM3 was efficiently and specifically phosphorylated by DAPk on a unique site, Ser160. Significantly DAPk phosphorylated this site in vivo upon overexpression in 293T cells. Activation of endogenous DAPk by increasing intracellular Ca2+ also led to increased phosphorylation of MCM3. Importantly short hairpin RNA-mediated knockdown of endogenous DAPk blocked both basal phosphorylation and Ca2+-induced phosphorylation, indicating that DAPk is both necessary and sufficient for MCM3 Ser160 phosphorylation in vivo. Identification of MCM3 as an in vivo DAPk substrate indicates the usefulness of this approach for identification of physiologically relevant substrates that may shed light on novel functions of the kinase.

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.