ZIP Kinase Research Articles

Novel ZIP kinase isoform lacks leucine zipper

Zipper-interacting protein kinase (ZIP kinase) has been thought to be involved in apoptosis and the C-terminal leucine zipper motif is important for its function. Recent studies have revealed that ZIP kinase also plays a role in regulating myosin phosphorylation. Here, we found novel ZIP kinase isoform in which the C-terminal non-kinase domain containing a leucine zipper is eliminated (hZIPK-S). hZIPK-S binds to myosin phosphatase targeting subunit 1(MYPT1) similar to the long isoform (hZIPK-L). In addition, we found that hZIPK-S as well as hZIPK-L bind to myosin. These results indicate that a leucine zipper is not critical for the binding of ZIP kinase to MYPT1 and myosin. Consistently, hZIPK-S localized with stress-fibers where they co-localized with myosin. The residues 278–311, the C-terminal side of the kinase domain common to the both isoforms, is involved in the binding to MYPT1, while the myosin binding domain is within the kinase domain. These results suggest that the newly found hZIPK-S as well as the long isoform play an important role in the regulation of myosin phosphorylation.

SIGNALING FOR CONTRACTION AND RELAXATION IN SMOOTH MUSCLE OF THE GUT

Phosphorylation of Ser19 on the 20-kDa regulatory light chain of myosin II (MLC20) by Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK) is essential for initiation of smooth muscle contraction. The initial [Ca2+]i transient is rapidly dissipated and MLCK inactivated, whereas MLC20 and muscle contraction are well maintained. Sustained contraction does not reflect Ca2+ sensitization because complete inhibition of MLC phosphatase activity in the absence of Ca2+ induces smooth muscle contraction. This contraction is suppressed by staurosporine, implying participation of a Ca2+-independent MLCK. Thus, sustained contraction, as with agonist-induced contraction at experimentally fixed Ca2+ concentrations, involves (a) G protein activation, (b) regulated inhibition of MLC phosphatase, and (c) MLC20 phosphorylation via a Ca2+-independent MLCK. The pathways that lead to inhibition of MLC phosphatase by G(q/13)-coupled receptors are initiated by sequential activation of Galpha(q)/alpha13, RhoGEF, and RhoA, and involve Rho kinase-mediated phosphorylation of the regulatory subunit of MLC phosphatase (MYPT1) and/or PKC-mediated phosphorylation of CPI-17, an endogenous inhibitor of MLC phosphatase. Sustained MLC20 phosphorylation is probably induced by the Ca2+-independent MLCK, ZIP kinase. The pathways initiated by G(i)-coupled receptors involve sequential activation of Gbetagamma(i), PI 3-kinase, and the Ca2+-independent MLCK, integrin-linked kinase. The last phosphorylates MLC20 directly and inhibits MLC phosphatase by phosphorylating CPI-17. PKA and PKG, which mediate relaxation, act upstream to desensitize the receptors (VPAC2 and NPR-C), inhibit adenylyl and guanylyl cyclase activities, and stimulate cAMP-specific PDE3 and PDE4 and cGMP-specific PDE5 activities. These kinases also act downstream to inhibit (a) initial contraction by inhibiting Ca2+ mobilization and (b) sustained contraction by inhibiting RhoA and targets downstream of RhoA. This increases MLC phosphatase activity and induces MLC20 dephosphorylation and muscle relaxation.

Physical and functional interactions between STAT3 and ZIP kinase

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors. It plays important roles in cell growth, apoptosis and cell transformation, and is constitutively active in a variety of tumor cells. In this study, we provide evidence that zipper-interacting protein kinase (ZIPK) interacts physically with STAT3. ZIPK specifically interacted with STAT3, and did not bind to STAT1, STAT4, STAT5a, STAT5b or STAT6. ZIPK phosphorylated STAT3 on serine 727 (Ser727) and enhanced STAT3 transcriptional activity. Small interfering RNA-mediated reduction of ZIPK expression decreased leukemia inhibitory factor (LIF)- and IL-6-induced STAT3-dependent transcription. Furthermore, LIF- and IL-6-mediated STAT3 activation stimulated ZIPK activity. Taken together, our data suggest that ZIPK interacts with STAT3 within the nucleus to regulate the transcriptional activity of STAT3 via phosphorylation of Ser727.

TCP10L is expressed specifically in spermatogenic cells and binds to death associated protein kinase‐3

The human transcriptional factor T-complex protein 10 like (TCP10L) gene is expressed exclusively in the liver and testis. However, the function of TCP10L in the testis remains unknown. We examined the expression of TCP10L in human testis and found that TCP10L was expressed specifically in the nucleus of spermatogenic cells during spermatogenesis. In addition, we identified death associated protein kinase 3 (DAPK-3/ZIP kinase) as a binding partner for TCP10L by yeast two-hybrid screening, followed with immunoprecipitation and subcellular localization experiments. Mutagenesis study revealed that this interaction was dependant on the leucine zipper motif-containing region. The specific expression pattern of TCP10L and interaction with DAPK-3 implies that TCP10L might play crucially important roles in spermatogenesis through its interaction with DAPK-3.

Daxx overexpression in T-lymphoblastic Jurkat cells enhances caspase-dependent death receptor- and drug-induced apoptosis in distinct ways

The role of Daxx, in particular, its ability to promote or hinder apoptosis, still remains controversial. In order to elucidate the functional relevance of Daxx in apoptosis signaling of malignant lymphocytes, Jurkat T-cells were stably transfected with a Daxx-expressing vector or with the respective Daxx-negative control vector. We thus demonstrate that ectopic expression of Daxx substantially increases the rate of apoptosis upon incubation with death receptor agonists such as Fas and TRAIL as well as upon incubation with the cytotoxic drug doxorubicin (DOX). Analysis of the molecular changes induced in the extrinsic and intrinsic apoptosis pathways reveals that augmentation of apoptosis by Daxx overexpression is conveyed by distinctly different mechanisms. Although enforced apoptosis caused by ectopic Daxx expression is caspase-dependent in both cases, major differences between Fas/TRAIL-induced apoptosis and doxorubicin-induced apoptosis are observed in expression patterns of X-linked inhibitor of apoptosis (XIAP), p53, Bid, ZIP kinase, and prostate apoptosis response gene 4 (Par-4). Moreover, we could show that addition of a CD95 blocking antibody to the clones treated with doxorubicin was able to increase apoptosis as compared to doxorubicin treatment alone and was accompanied by an enhancement of the mitochondrial branch of apoptosis. In conclusion, we here outline the major molecular mechanisms underlying the apoptosis-promoting effect of Daxx in neoplastic lymphocytes and demonstrate fundamental molecular differences elicited by the overexpression of Daxx in the extrinsic and intrinsic signaling pathways.

Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions.

The death-associated protein (DAP) kinase family includes three protein kinases, DAP kinase, DAP kinase-related protein 1, and ZIP kinase, which display 80% amino acid identity within their catalytic domains and are functionally linked to common subcellular changes occurring during cell death, such as the process of membrane blebbing. Here we show physical and functional cross talk between DAP kinase and ZIP kinase. The two kinases display strong synergistic effects on cell death when coexpressed and physically bind each other via their catalytic domains. Furthermore, DAP kinase phosphorylates ZIP kinase at six specific sites within its extracatalytic C-terminal domain. ZIP kinase localizes to both the nucleus and the cytoplasm and fractionates as monomeric and trimeric forms. Significantly, modification of the DAP kinase phosphorylation sites influences both the localization and oligomerization status of ZIP kinase. A mutant ZIP kinase construct, in which the six serine/threonine residues were mutated to aspartic acid to mimic the phosphorylated state, was found predominantly in the cytoplasm as a trimer and possessed greater cell death-inducing potency. This suggests that DAP kinase and ZIP kinase function in a biochemical pathway in which DAP kinase activates the cellular function of ZIP kinase through phosphorylation, leading to amplification of death-promoting signals.

Reduced Expression of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) in Squamous Cell Carcinoma Complicating Recessive Dystrophic Epidermolysis Bullosa

Squamous cell carcinoma (SCC) is a common complication in individuals with recessive dystrophic epidermolysis bullosa (RDEB). For the severe Hallopeau-Siemens subtype, the mortality rate from SCC is over 55% by the age of 40 y. Currently, little is known about the molecular pathology or cell biology of SCC in RDEB. In this study, we compared gene expression in RDEB SCC (n=3) and non-EB SCC (n=3) with corresponding RDEB and non-EB peri-tumoral skin, with microarray analysis using DermArray membranes as well as semi-quantitative and real-time RT-PCR. Both tumor sets showed downregulation of epidermal differentiation markers (e.g., profilaggrin, keratins 1 and 10) as well as certain pro-apoptotic genes (e.g., death-associated kinase-3 or ZIP kinase). Likewise, in both groups there was upregulation of matrix metalloproteinase 1 and laminin 5 in the tumors. But we found that the expression of insulin-like growth factor-binding protein-3 (IGFBP-3) was lower (mean of 5.8-fold) in RDEB SCC compared with non-EB SCC. These data were verified by immunohistochemistry. IGFBP-3 has an important role in cancer cell apoptosis mediated via the nuclear retinoid X receptor alpha (RXRalpha). Reduced expression of IGFBP-3 in RDEB SCC may provide a partial explanation for the aggressive behavior and poor prognosis of these tumors in this genodermatosis.

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 p21WAF1. ZIP kinase phosphorylates p21WAF1 at Thr145 and alanine-substituted mutations in the p21WAF1 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 p21WAF1 at Thr145 in vivo and can increase the half-life of p21WAF1, while the half-life of p21WAF1[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 p21WAF1, and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.

ZIP kinase triggers apoptosis from nuclear PML oncogenic domains.

PML oncogenic domains (PODs), also referred to as nuclear dot 10 bodies, Kreb's bodies, or nuclear bodies, represent nuclear structures implicated in the regulation of a variety of cellular processes, including transcription, tumor suppression, and apoptosis. ZIP kinase (ZIPK) is a proapoptotic protein kinase with homology to DAP kinase, a protein kinase implicated in apoptosis. We show here that ZIPK is present in PODs, where it colocalizes with and binds to proapoptotic protein Daxx. Arsenic trioxide (As(2)O(3)) and gamma interferon (IFN-gamma), which accentuate POD formation, increased the association of ZIPK with PODs. In contrast, the kinase-inactive ZIPK resides in nuclei with a diffuse pattern and significantly prevents the association of Daxx with PODs, implying that ZIPK recruits Daxx to PODs via its catalytic activity. ZIPK also binds and phosphorylates proapoptotic protein Par-4. Association of ZIPK with Daxx was enhanced by coexpression of Par-4. Activation of caspases and induction of apoptosis were also observed in cells overexpressing these proteins. Conversely, small-interfering RNA-mediated reduction of ZIPK, Daxx, or Par-4 expression decreased activation of caspase and apoptosis induced by As(2)O(3) and IFN-gamma. These results suggest that ZIPK, in collaboration with Daxx and Par-4, mediates a novel nuclear pathway for apoptosis.

Gene array analysis of bone morphogenetic protein type I receptor-induced osteoblast differentiation.

The genomic response to BMP was investigated by ectopic expression of activated BMP type I receptors in C2C12 myoblast using cDNA microarrays. Novel BMP receptor target genes with possible roles in inhibition of myoblast differentiation and stimulation of osteoblast differentiation were identified. Bone morphogenetic proteins (BMPs) have an important role in controlling mesenchymal cell fate and mediate these effects by regulating gene expression. BMPs signal through three distinct specific BMP type I receptors (also termed activin receptor-like kinases) and their downstream nuclear effectors, termed Smads. The critical target genes by which activated BMP receptors mediate change cell fate are poorly characterized. We performed transcriptional profiling of C2C12 myoblasts differentiation into osteoblast-like cells by ectopic expression of three distinct constitutively active (ca)BMP type I receptors using adenoviral gene transfer. Cells were harvested 48 h after infection, which allowed detection of both early and late response genes. Expression analysis was performed using the mouse GEM1 microarray, which is comprised of approximately 8700 unique sequences. Hybridizations were performed in duplicate with a reverse fluor labeling. Genes were considered to be significantly regulated if the p value for differential expression was less than 0.01 and inverted expression ratios per duplicate successful reciprocal hybridizations differed by less than 25%. Each of the three caBMP type I receptors stimulated equal levels of R-Smad phosphorylation and alkaline phosphatase activity, an early marker for osteoblast differentiation. Interestingly, all three type I receptors induced identical transcriptional profiles; 97 genes were significantly upregulated and 103 genes were downregulated. Many extracellular matrix genes were upregulated, muscle-related genes downregulated, and transcription factors/signaling components modulated. In addition to 41 expressed sequence tags without known function and a number of known BMP target genes, including PPAR-gamma and fibromodulin, a large number of novel BMP target genes with an annotated function were identified, including transcription factors HesR1, ITF-2, and ICSBP, apoptosis mediators DRP-1 death kinase and ZIP kinase, IkappaB alpha, Edg-2, ZO-1, and E3 ligase Dactylin. These target genes, some of them unexpected, offer new insights into how BMPs elicit biological effects, in particular into the mechanism of inhibition of myoblast differentiation and stimulation of osteoblast differentiation.

Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch.

Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 microM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.

Regulation of smooth muscle contraction by calcium, monomeric GTPases of the Rho subfamily and their effector kinases.

A key event in the activation of smooth muscle contraction ist the phosphorylation of the regulatory light chains of myosin (r-MLC) at Ser19 which is predominantly catalyzed by the Ca2+ and calmodulin dependent myosin light chain kinase, MLCK (Gallager et al., 1997). Recently, it has been suggested that, in addition, r-MLC phosphorylation and contraction may be induced in a Ca2+-independent manner by several protein kinases, such as Rho associated kinase, ZIP kinase, and integrin linked kinase, ILK (reviewed in Ganitkevich et al, 2002). These kinases may, hence, be involved in Ca2+-independent contractions (Kureishi et al., 1999) leading to an increased Ca2+-sensitivity of r-MLC phosphorylation and contraction. Although the physiological role of these kinases is far from clear, they may be of importance during the maintained phase of a contraction when intracellular [Ca2+] has returned to near resting values (Himpens and Somlyo, 1988; Lucius et al., 1998). One goal of this study, therefore, was to test whether Ca2+ is required for tension maintenance of an agonist induced contraction using the membrane permeant form of the caged Ca-chelator, diazo2.

DAP-like kinase interacts with the rat homolog of Schizosaccharomyces pombe CDC5 protein, a factor involved in pre-mRNA splicing and required for G2/M phase transition

DAP-like kinase (Dlk, also termed ZIP kinase) is a leucine zipper-containing serine/threonine-specific protein kinase with as yet unknown biological function(s). Interaction partners so far identified are either transcription factors or proteins that can support or counteract apoptosis. Thus, Dlk might be involved in regulating transcription or, more generally, survival or apoptosis. Here we report on a new interaction partner, the rat homolog of Schizosaccharomyces pombe CDC5 protein, a presumptive transcription and splicing factor involved in the G(2)/M transition. In vitro, rat CDC5 forms complexes with, but is not phosphorylated by, Dlk. Rather, it was phosphorylated by an associated kinase which was identified as CK2. The interaction domain of Dlk was mapped to the leucine zipper, while that of CDC5 was mapped to the C-terminal region between residues 500 and 802. In vivo, both proteins co-localize perfectly in distinct speckle-like structures in the nucleus, some of which overlap with promyelocytic leukemia protein. Interestingly, splicing factor SC35, which also resides in speckles, was partially displaced upon overexpression of either CDC5 or Dlk, perhaps due to phosphorylation by Dlk. Together with previous data, these results suggest that Dlk might play a role in coordinating specific transcription and splicing events.

HeLa ZIP kinase induces diphosphorylation of myosin II regulatory light chain and reorganization of actin filaments in nonmuscle cells.

Dlk/ZIP kinase is a serine/threonine kinase highly homologous to DAP kinase. We have reported that HeLa ZIP kinase (hZIPK) phosphorylated the regulatory light chain of myosin II (MRLC) at both Ser19 and Thr18 in vitro. In this study, we demonstrate that hZIPK also induces the diphosphorylation of MRLC in nonmuscle cells. Peptide mapping revealed that transient transfection of hZIPK into HeLa cells caused diphosphorylation of MRLC. In contrast, transfection of the kinase inactive mutant of hZIPK did not induce any phosphorylation of MRLC. Using antibodies specific for mono- or diphosphorylated MRLC, we showed that diphosphorylated MRLC induced by the overexpression of hZIPK was concentrated in striking aggregates or bundles of actin filaments in HeLa cells, while monophosphorylated MRLC showed no prominent localization to these aggregates. Overexpression of hZIPK also induced dramatic changes in cell shape and disruption of nuclear morphology reminiscent of changes during apoptosis. These effects of hZIPK were suppressed by the coexpression of a mutant MRLC where both phosphorylation sites were replaced with alanine, indicating that the changes in actin organization were a consequence of MRLC diphosphorylation. These results suggested that hZIPK plays a role in regulating actin organization and cell morphology in non-muscles and at least part of its effects are mediated through the diphosphorylation of MRLC.

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with 32P release and a point mutant of Thr 38.

Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis.

In this study we describe the identification and structure-function analysis of a novel death-associated protein (DAP) kinase-related protein, DRP-1. DRP-1 is a 42-kDa Ca(2+)/calmodulin (CaM)-regulated serine threonine kinase which shows high degree of homology to DAP kinase. The region of homology spans the catalytic domain and the CaM-regulatory region, whereas the remaining C-terminal part of the protein differs completely from DAP kinase and displays no homology to any known protein. The catalytic domain is also homologous to the recently identified ZIP kinase and to a lesser extent to the catalytic domains of DRAK1 and -2. Thus, DAP kinase DRP-1, ZIP kinase, and DRAK1/2 together form a novel subfamily of serine/threonine kinases. DRP-1 is localized to the cytoplasm, as shown by immunostaining and cellular fractionation assays. It binds to CaM, undergoes autophosphorylation, and phosphorylates an exogenous substrate, the myosin light chain, in a Ca(2+)/CaM-dependent manner. The truncated protein, deleted of the CaM-regulatory domain, was converted into a constitutively active kinase. Ectopically expressed DRP-1 induced apoptosis in various types of cells. Cell killing by DRP-1 was dependent on two features: the status of the catalytic activity, and the presence of the C-terminal 40 amino acids shown to be required for self-dimerization of the kinase. Interestingly, further deletion of the CaM-regulatory region could override the indispensable role of the C-terminal tail in apoptosis and generated a "superkiller" mutant. A dominant negative fragment of DAP kinase encompassing the death domain was found to block apoptosis induced by DRP-1. Conversely, a catalytically inactive mutant of DRP-1, which functioned in a dominant negative manner, was significantly less effective in blocking cell death induced by DAP kinase. Possible functional connections between DAP kinase and DRP-1 are discussed.

Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis.

Dlk/ZIP kinase is a newly discovered serine/threonine kinase which, due to its homology to DAP kinase, was named DAP like kinase, Dlk. This kinase is tightly associated with nuclear structures, it undergoes extensive autophosphorylation and phosphorylates myosin light chain and core histones H3, H2A and H4 in vitro. Moreover, it possesses a leucine zipper which mediates interaction with transcription factor ATF-4, therefore it was called ZIP kinase. We employed the yeast two-hybrid system to identify interaction partners of Dlk that might serve as regulators or targets. Besides ATF-4 and others we found Par-4, a modulator of transcription factor WT1 and mediator of apoptosis. Complex formation between Dlk and Par-4 was confirmed by GST pull-down experiments and kinase reactions in vitro and coexpression experiments in vivo. The interaction domain within Dlk was mapped to an arginine-rich region between residues 338 - 417, rather than to the leucine zipper. Strikingly, coexpression of Dlk and Par-4 lead to relocation of Dlk from the nucleus to the cytoplasm, particularly to actin filaments. These interactions provoked a dramatic reorganization of the cytoskeleton and morphological symptoms of apoptosis, thus suggesting a functional relationship between Dlk and Par-4 in the control of apoptosis.

C-terminal truncation of Dlk/ZIP kinase leads to abrogation of nuclear transport and high apoptotic activity.

Dlk (also termed ZIP kinase) is a novel serine/threonine kinase with a unique C-terminal domain that is rich in arginine and contains three putative NLS motifs and a functional lecuine zipper. Dlk is indeed localized in the nucleus where it shows a speckled distribution. To elucidate the biological functions of Dlk, we wanted to identify the signals relevant for nuclear transport and further the nuclear structures which Dlk binds to. Expression of various deletion and point mutations of Dlk as GFP fusion proteins revealed that the leucine zipper is required for association with speckles and the most C-terminal NLS is necessary and sufficient for nuclear transport. Interestingly, a C-terminal deletion mutant defective for nuclear transport exhibited a pronounced colocalization with actin filaments and, even more strikingly, was a very potent inducer of apoptosis. This apoptotic activity was abrogated, however, when this mutant was retargeted to the nucleus via a heterologous NLS from large T, indicating that Dlk only exerts an apoptotic activity in the cytoplasm. To identify the speckle like structures to which Dlk binds we performed immunofluorescence analyses with antibodies directed against representative marker proteins of replication, transcription, or splicing centers. None of these marker proteins revealed a colocalization with Dlk. Instead, we found a partial colocalization with PML bodies which seem to play a key role in regulation of apoptosis. Taken together, these data strongly suggest a functional role for Dlk in control of cell survival which is dependent on its subcellular localization.

AATF, a novel transcription factor that interacts with Dlk/ZIP kinase and interferes with apoptosis

Dlk, also known as ZIP kinase, is a serine/threonine kinase that is tightly associated with nuclear structures. Under certain conditions, which require cytoplasmic localization, Dlk can induce apoptosis. In search for interaction partners that might serve as regulators or targets of this kinase we identified apoptosis antagonizing transcription factor (AATF), a nuclear phosphoprotein of 523 amino acids. The 1.8 kb mRNA seems to be ubiquitously expressed. AATF contains an extremely acidic domain and a putative leucine zipper characteristic of transcription factors. Indeed, a Gal4-BD-AATF fusion protein exhibited strong transactivation activity. Interestingly, AATF interfered with Dlk-induced apoptosis.

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642–1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca 2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin-activated MgATPase activity of myosin II. HeLa ZIPK is the first non-muscle MLCK that phosphorylates MRLC at two sites.