DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation

Bialik, Shani; Bresnick, Anne R.; Kimchi, Adi

doi:10.1038/sj.cdd.4401386

Cited by 82 publications

(92 citation statements)

References 43 publications

(69 reference statements)

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“…This decrease indicated a significant average of 19 ± 5% increase in doubling time (Figure 4d; Po0.04, paired Student's t-test). As expected (Bialik et al, 2004), DAPk lacking the catalytic domain did not induce cell death in these experiments as measured by Trypan blue staining.…”

Section: Dapk Regulates Cell Metabolism Through Pkm2supporting

confidence: 85%

“…To determine whether DAPk can promote PKM2 activation in cells independent of its catalytic activity, HEK293T and HeLa JW subline (described in Bialik et al, 2004) were transfected with truncated DAPk lacking the KD (DAPkDKD), or with a structurally irrelevant protein. These cell lines express very low levels of endogenous DAPk, which allowed us to determine the direct effects of the different ectopically expressed mutant proteins.…”

Section: Ectopic Expression Of Catalytically Inactive Dapk Enhances Ementioning

confidence: 99%

“…I Mor et al is responsible for its binding to the cytoskeleton (Figure 1a), a considerable portion of DAPk in cells is tightly bound to the actin microfilaments (Bialik et al, 2004). Therefore, to disrupt actin filaments and gently solubilize cytoskeletal protein complexes, cells were incubated with latrunculin-B before protein extraction.…”

Section: Dapk Regulates Cell Metabolism Through Pkm2mentioning

confidence: 99%

“…It has since been characterized as a tumor suppressor protein whose expression is lost in a significant number of human malignancies (Bialik et al, 2004;Gozuacik and Kimchi, 2006;Bialik and Kimchi, 2010). Loss of DAPk expression is frequently caused by epigenetic silencing through promoter hypermethylation.…”

Section: Introductionmentioning

confidence: 99%

“…The multi-domain structure of DAPk also includes a stretch of ankyrin repeats, and a cytoskeleton-binding region, which localize the protein to the actin microfilaments (Cohen et al, 1997;Bialik et al, 2004) (Figure 1a). Near its C terminus, DAPk harbors a death domain, a six-helical protein-protein interaction module found in various apoptotic proteins Raveh et al, 2000;Goldschneider and Mehlen, 2010).…”

Section: Introductionmentioning

confidence: 99%

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Death-associated protein kinase increases glycolytic rate through binding and activation of pyruvate kinase

et al. 2011

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Death-associated protein kinase (DAPk), a multi-domain serine/threonine kinase, regulates numerous cell death mechanisms and harbors tumor suppressor functions. In this study, we report that DAPk directly binds and functionally activates pyruvate kinase M2 (PKM2), a key glycolytic enzyme, which contributes to the regulation of cancer cell metabolism. PKM2 was identified as a novel binding partner of DAPk by a yeast two-hybrid screen. This interaction was validated in vitro by enzyme-linked immunosorbent assay using purified proteins and in vivo by co-immunoprecipitation of the two endogenous proteins from cells. In vitro interaction with full-length DAPk resulted in a significant increase in the activity of PKM2. Conversely, a fragment of DAPk harboring only the functional kinase domain (KD) could neither bind PKM2 in cells nor activate it in vitro. Indeed, DAPk failed to phosphorylate PKM2. Notably, transfection of cells, with a truncated DAPk lacking the KD, elevated endogenous PKM2 activity, suggesting that PKM2 activation by DAPk occurs independently of its kinase activity. DAPk-transfected cells displayed changes in glycolytic activity, as reflected by elevated lactate production, whereas glucose uptake remained unaltered. A mild reduction in cell proliferation was detected as well in these transfected cells. Altogether, this work identifies a new role for DAPk as a metabolic regulator, suggesting the concept of direct interactions between a tumor suppressor and a key glycolytic enzyme to limit cell growth. Moreover, the work documents a unique function of DAPk that is independent of its catalytic activity and a novel mechanism to activate PKM2 by protein-protein interaction.

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Section: Dapk Regulates Cell Metabolism Through Pkm2supporting

confidence: 85%

Section: Ectopic Expression Of Catalytically Inactive Dapk Enhances Ementioning

confidence: 99%

Section: Dapk Regulates Cell Metabolism Through Pkm2mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Death-associated protein kinase increases glycolytic rate through binding and activation of pyruvate kinase

et al. 2011

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Shear stress regulates expression of death‐associated protein kinase in suppressing TNFα‐induced endothelial apoptosis

Rennier

2012

Journal Cellular Physiology

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Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)-induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto-inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm(2) for 6 h. Quantitative RT-PCR revealed a corresponding increase in DAPK mRNA [P < 0.01]. We found that after 18-h TNFα induction, shearing cells for another 6 h significantly reduced apoptosis based on TUNEL staining [P < 0.05], although cell necrosis was not affected. Under the same conditions, we observed significantly decreased overall DAPK, as well as phospho-serine 308 DAPK [P < 0.05] compared to TNFα treatment alone. Caspase-3 and -7 activities downstream of DAPK were also attenuated. Shearing cells alone resulted in enhanced apoptosis, likely due to increased DAPK activity. Our findings were further supported by DAPK siRNA, which yielded contrary results. We present conclusive evidence for the first time that shear stress of up to 6 h up-regulates DAPK expression and activation. However, in the presence of apoptotic stimuli such as TNFα, shear stress caused decrease in DAPK activity. In fact, long-term shear stress of 24 h significantly reduced overall DAPK expression. Our findings strongly support a novel role for DAPK in mediating effects of shear stress in suppressing cytokine-activated apoptosis.

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The Roc domain of leucine‐rich repeat kinase 2 is sufficient for interaction with microtubules

Gandhi

Wang

Zhu

et al. 2008

J of Neuroscience Research

155

142

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Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson's disease (PD). While this multi-domain protein has been shown to have both GTPase and kinase activities through the Roc and MAPKKK domains, respectively, the proteinprotein interactions and pathways involved in LRRK2-mediated signaling remain elusive. Utilizing a combination of protein pull-down assays, mass spectrometry, Western blotting and immunofluorescence microscopy, this study identifies and describes the interaction between LRRK2 and microtubules. The Roc or GTPase-like domain of LRRK2 is sufficient for interaction with α/β-tubulin heterodimers. This interaction occurs in a guanine-nucleotide independent manner, suggesting that tubulin may not be an effector of the LRRK2 GTPase domain. The R1441C pathogenic mutation, located within the Roc domain, retains interaction with α/β-tubulin heterodimers, suggesting that disruption of this interaction is not likely the mechanism whereby the R1441C mutation leads to disease. At a subcellular level, endogenous LRRK2 protein was found to colocalize with α/β-tubulin in primary hippocampal neurons. These findings are significant because they link LRRK2 with microtubules, a structural component of the cell that is critically involved in the pathogenesis of several neurodegenerative diseases, including PD.

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DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation

Cited by 82 publications

References 43 publications

Death-associated protein kinase increases glycolytic rate through binding and activation of pyruvate kinase

Death-associated protein kinase increases glycolytic rate through binding and activation of pyruvate kinase

Shear stress regulates expression of death‐associated protein kinase in suppressing TNFα‐induced endothelial apoptosis

The Roc domain of leucine‐rich repeat kinase 2 is sufficient for interaction with microtubules

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