Death-Associated Protein Kinase-Related Protein 1, a Novel Serine/Threonine Kinase Involved in Apoptosis

ABSTRACT 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 Ca2+/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 Ca2+/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.

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Dap-Kinase Participates in TNF-α–And FAS-Induced Apoptosis and Its Function Requires the Death Domain

The Journal of Cell Biology ◽

10.1083/jcb.146.1.141 ◽

1999 ◽

Vol 146 (1) ◽

pp. 141-148 ◽

Cited By ~ 48

Author(s):

Ofer Cohen ◽

Boaz Inbal ◽

Joseph L. Kissil ◽

Tal Raveh ◽

Hanna Berissi ◽

...

Keyword(s):

Cell Death ◽

Dominant Negative ◽

Protein Domain ◽

Dominant Negative Mutant ◽

Death Domain ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Dap Kinase ◽

Tnf Α ◽

Induced Apoptosis

Death-associated protein (DAP)–kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-α and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti–Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-α, Fas, and FADD/MORT1–induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.

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Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6286 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6286-6296 ◽

Cited By ~ 257

Author(s):

Tadahiro Kitamura ◽

Yukari Kitamura ◽

Shoji Kuroda ◽

Yasuhisa Hino ◽

Miwa Ando ◽

...

Keyword(s):

Cyclic Nucleotide ◽

Second Messengers ◽

Dominant Negative ◽

Cyclic Nucleotide Phosphodiesterase ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Serine Threonine Kinase ◽

Intact Cells ◽

Threonine Kinase

ABSTRACT Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.

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Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

Biochemical Journal ◽

10.1042/bj20100799 ◽

2010 ◽

Vol 432 (1) ◽

pp. 77-87 ◽

Cited By ~ 12

Author(s):

Joanne Durgan ◽

Peter J. Parker

Keyword(s):

Mammalian Cells ◽

Tumour Suppressor ◽

Dependent Manner ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Pkc Protein Kinase C ◽

Specific Regulation ◽

The Relationship ◽

Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

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Crystal structure of microtubule affinity-regulating kinase 4 catalytic domain in complex with a pyrazolopyrimidine inhibitor

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15024747 ◽

2016 ◽

Vol 72 (2) ◽

pp. 129-134 ◽

Cited By ~ 22

Author(s):

John S. Sack ◽

Mian Gao ◽

Susan E. Kiefer ◽

Joseph E. Myers ◽

John A. Newitt ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Neurofibrillary Tangle ◽

Binding Pocket ◽

Atp Binding ◽

Activation Loop ◽

Serine Threonine Kinase ◽

Atp Binding Site ◽

Threonine Kinase ◽

Catalytic Loop

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulate microtubule dynamics. Abnormal activity of MARK4 has been proposed to contribute to neurofibrillary tangle formation in Alzheimer's disease. The crystal structure of the catalytic and ubiquitin-associated domains of MARK4 with a potent pyrazolopyrimidine inhibitor has been determined to 2.8 Å resolution with anRworkof 22.8%. The overall structure of MARK4 is similar to those of the other known MARK isoforms. The inhibitor is located in the ATP-binding site, with the pyrazolopyrimidine group interacting with the inter-lobe hinge region while the aminocyclohexane moiety interacts with the catalytic loop and the DFG motif, forcing the activation loop out of the ATP-binding pocket.

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Neuronal Cell Shape and Neurite Initiation Are Regulated by the Ndr Kinase SAX-1, a Member of the Orb6/COT-1/Warts Serine/Threonine Kinase Family

Molecular Biology of the Cell ◽

10.1091/mbc.11.9.3177 ◽

2000 ◽

Vol 11 (9) ◽

pp. 3177-3190 ◽

Cited By ~ 63

Author(s):

Jennifer A. Zallen ◽

Erin L. Peckol ◽

David M. Tobin ◽

Cornelia I. Bargmann

Keyword(s):

Cell Shape ◽

Neuronal Cell ◽

Neurite Growth ◽

Dominant Negative ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

C Elegans ◽

Calcium Calmodulin Dependent ◽

Binding Domains ◽

Neurite Initiation

The Caenorhabditis elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those ofsax-1 mutants, and genetic interactions betweenrhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading.

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Saccharomyces cerevisiae Env7 is a novel serine/threonine kinase 16 (STK16)‐related protein kinase and negatively regulates organelle fusion at the lysosomal vacuole

The FASEB Journal ◽

10.1096/fasebj.27.1_supplement.1016.2 ◽

2013 ◽

Vol 27 (S1) ◽

Author(s):

Editte Gharakhanian ◽

Surya Manandhar ◽

Florante Ricarte ◽

Stephanie Cocca

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Related Protein ◽

Serine Threonine Kinase ◽

Threonine Kinase

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Mothers against dpp participates in a DDP/TGF-beta responsive serine-threonine kinase signal transduction cascade

Development ◽

10.1242/dev.124.16.3167 ◽

1997 ◽

Vol 124 (16) ◽

pp. 3167-3176 ◽

Cited By ~ 14

Author(s):

S.J. Newfeld ◽

A. Mehra ◽

M.A. Singer ◽

J.L. Wrana ◽

L. Attisano ◽

...

Keyword(s):

Signal Transduction ◽

Nuclear Accumulation ◽

Target Cells ◽

Type I ◽

Dependent Manner ◽

Tgf Beta ◽

Serine Threonine Kinase ◽

Drosophila Cell ◽

Threonine Kinase ◽

Signal Transduction Cascade

Mothers against dpp (Mad) is the prototype of a family of genes required for signaling by TGF-beta related ligands. In Drosophila, Mad is specifically required in cells responding to Decapentaplegic (DPP) signals. We further specify the role of Mad in DPP-mediated signaling by utilizing tkvQ199D, an activated form of the DPP type I receptor serine-threonine kinase thick veins (tkv). In the embryonic midgut, tkvQ199D mimics DPP-mediated inductive interactions. Homozygous Mad mutations block signaling by tkvQ199D. Appropriate responses to signaling by tkvQ199D are restored by expression of MAD protein in DPP-target cells. Endogenous MAD is phosphorylated in a ligand-dependent manner in Drosophila cell culture. DPP overexpression in the embryonic midgut induces MAD nuclear accumulation; after withdrawal of the overexpressed DPP signal, MAD is detected only in the cytoplasm. However, in three different tissues and developmental stages actively responding to endogenous DPP, MAD protein is detected in the cytoplasm but not in the nucleus. From these observations, we discuss possible roles for MAD in a DPP-dependent serine-threonine kinase signal transduction cascade integral to the proper interpretation of DPP signals.

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The serine/threonine kinase ULK1 is a target of multiple phosphorylation events

Biochemical Journal ◽

10.1042/bj20101894 ◽

2011 ◽

Vol 440 (2) ◽

pp. 283-291 ◽

Cited By ~ 133

Author(s):

Markus Bach ◽

Mark Larance ◽

David E. James ◽

Georg Ramm

Keyword(s):

Mammalian Target Of Rapamycin ◽

Phosphorylation Site ◽

Degradation Process ◽

Dominant Negative ◽

Serine Threonine Kinase ◽

Kinase Activation ◽

Threonine Kinase ◽

Convergence Point ◽

Mtor Phosphorylation

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser555in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser774 and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr180 that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.

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RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6325 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6325-6339 ◽

Cited By ~ 110

Author(s):

Wun-Chey Sin ◽

Xiang-Qun Chen ◽

Thomas Leung ◽

Louis Lim

Keyword(s):

Structural Integrity ◽

Morphological Changes ◽

Dominant Negative ◽

Cell Periphery ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Complex Process ◽

Rho Effectors ◽

Filament Network

ABSTRACT The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.

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