WHEP domains direct noncanonical function of glutamyl-Prolyl tRNA synthetase in translational control of gene expression

The heterotetrameric GAIT complex suppresses translation of selected mRNAs in interferon-gamma-activated monocytic cells. Specificity is dictated by glutamyl-prolyl tRNA synthetase (EPRS) binding to a 3'UTR element in target mRNAs. EPRS consists of two synthetase cores joined by a linker containing three WHEP domains of unknown function. Here we show the critical role of EPRS WHEP domains in targeting and regulating GAIT complex binding to RNA. The upstream WHEP pair directs high-affinity binding to GAIT element-bearing mRNAs, while the overlapping, downstream pair binds NSAP1, which inhibits mRNA binding. Interaction of EPRS with ribosomal protein L13a and GAPDH induces a conformational switch that rescues mRNA binding and restores translational control. Total reconstitution from purified components indicates that the four GAIT proteins are necessary and sufficient for self-assembly of a functional complex. Our results establish the essentiality of WHEP domains in the noncanonical function of EPRS in regulating inflammatory gene expression.     

Regulation of inflammation by DAPK

Death-associated protein kinase (DAPK) is a tumor suppressor and negatively regulates several activation signals. Consistent with its potential anti-inflammatory activity, DAPK promotes the formation of IFN-γ-activated inhibitor of translation (GAIT) complex that suppresses the translation of selected inflammatory genes. DAPK has been found to inhibit tumor necrosis factor-α (TNF-α)- or lipopolysaccharides (LPS)-induced NF-κB activation and pro-inflammatory cytokine expression. Inflammation is always associated with T cell activation, while DAPK attenuates T cell activation by a selective suppression in T cell receptor-triggered NF-κB activation. Recent studies, however, also reveal a contribution of DAPK to pro-inflammatory processes. DAPK is shown to mediate pro-inflammatory signaling downstream of TNF-α, LPS, IL-17, or IL-32. In addition, DAPK is required for the full formation of NLRP3 inflammasome, essential for the generation of IL-1β and IL-18. These results suggest the complicated role of DAPK in the regulation of inflammation that is likely dependent on cell types and environmental cues.     

Post-translational regulation of the cellular levels of DAPK

Death associated protein kinase (DAPK) is a large, multi-domain ser/thr kinase whose activities converge upon multiple signaling pathways that regulate autophagy, caspase-dependent cell death, cell adhesion and migration. The cellular levels of DAPK are post-translationally regulated by the combined activities of two degradation systems, including the ubiquitin proteasome and an extra-lysosomal proteolysis pathway. At least three distinct E3 ubiquitin ligases target DAPK, including mindbomb1, the chaperone dependent ligase, CHIP (carboxy terminus of Hsp70-interacting protein) and a cullin RING ligase complex, KLHL20-Cul3-RBX1. In addition, it appears that the cellular levels of DAPK are also regulated by an extra-lysosomal protease, cathepsin B. While protein quality control and recycling clearly benefit cells by removal of misfolded or toxic proteins and recycling of their components, the finding that multiple surveillance systems target DAPK suggests that these protein degradation systems also act to fine tune DAPK expression levels in response to specific signaling pathways.     

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.     

Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1

Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.     

The tumor suppressor death-associated protein kinase targets to TCR-stimulated NF-kappa B activation

Death-associated protein kinase (DAPK) is a unique multidomain kinase acting both as a tumor suppressor and an apoptosis inducer. The molecular mechanism underlying the effector function of DAPK is not fully understood, while the role of DAPK in T lymphocyte activation is mostly unknown. DAPK was activated after TCR stimulation. Through the expression of a dominant-negative and a constitutively active form of DAPK in T cells, we found that DAPK negatively regulated T cell activation. DAPK markedly affected T cell proliferation and IL-2 production. We identified TCR-induced NF-kappaB activation as a target of DAPK. In contrast, IL-1beta- and TNF-alpha-triggered NF-kappaB activation was not affected by DAPK. We further found that DAPK selectively modulated the TCR-induced translocation of protein kinase Ctheta, Bcl-10, and IkappaB kinase into membrane rafts. Notably, the effect of DAPK on the raft entry was specific for the NF-kappaB pathway, as other raft-associated molecules, such as linker for activation of T cells, were not affected. Our results clearly demonstrate that DAPK is a novel regulator targeted to TCR-activated NF-kappaB and T cell activation.     

ZIPK: a unique case of murine-specific divergence of a conserved vertebrate gene

Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function.     

The DAPK family: a structure–function analysis

DAP-kinase (DAPK) is the founding member of a family of highly related, death associated Ser/Thr kinases that belongs to the calmodulin (CaM)-regulated kinase superfamily. The family includes DRP-1 and ZIP-kinase (ZIPK), both of which share significant homology within the common N-terminal kinase domain, but differ in their extra-catalytic domains. Both DAPK and DRP-1 possess a conserved CaM autoregulatory domain, and are regulated by calcium-activated CaM and by an inhibitory auto-phosphorylation within the domain. ZIPK’s activity is independent of CaM but can be activated by DAPK. The three kinases share some common functions and substrates, such as induction of autophagy and phosphorylation of myosin regulatory light chain leading to membrane blebbing. Furthermore, all can function as tumor suppressors. However, they also each possess unique functions and intracellular localizations, which may arise from the divergence in structure in their respective C-termini. In this review we will introduce the DAPK family, and present a structure/function analysis for each individual member, and for the family as a whole. Emphasis will be placed on the various domains, and how they mediate interactions with additional proteins and/or regulation of kinase function.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

Zipper interacting protein kinase (ZIPK): function and signaling

Zipper interacting protein kinase (ZIPK), also known as death associated protein kinase 3, is a serine/threonine kinase that mediates variety of cell functions. The major biologic function of ZIPK is considered to be the regulation of apoptosis and smooth muscle contraction. Recently, several other functions of ZIPK have been gradually clarified. In this review article, we summarized the recent findings on ZIPK function and ZIPK-related cell signaling. We propose that ZIPK is a potential future target for the development of pharmaceutical therapy for cancer as well as cardiovascular diseases.     

Protection of Extraribosomal RPL13a by GAPDH and Dysregulation by S-Nitrosylation

Multiple eukaryotic ribosomal proteins (RPs) are co-opted for extraribosomal "moonlighting" activities, but paradoxically, RPs exhibit rapid turnover when not ribosome-bound. In one illustrative case of a functional extraribosomal RP, interferon (IFN)-γ induces ribosome release of L13a and assembly into the IFN-gamma-activated inhibitor of translation (GAIT) complex for translational control of a subset of?inflammation-related proteins. Here we show GAPDH functions as a chaperone, shielding newly released L13a from proteasomal degradation. However, GAPDH protective activity is lost following cell?treatment with oxidatively modified low density lipoprotein and IFN-γ. These agonists stimulate S-nitrosylation at Cys(247) of GAPDH, which fails to interact with L13a, causing proteasomal degradation of essentially the entire cell complement of L13a and defective translational control. Evolution of extraribosomal RP activities might require coevolution of?protective chaperones, and pathological disruption of either protein, or their interaction, presents an alternative mechanism of diseases due to RP defects, and targets for therapeutic intervention.