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Christian Doerig - One of the best experts on this subject based on the ideXlab platform.
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A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-Related Kinase
Malaria journal, 2016Co-Authors: Deborah Mitcheson, Andrew M. Fry, Christian Doerig, Andrew R. Bottrill, Katherine H. Carr, Christopher R. Coxon, Celine Cano, Bernard T. Golding, Roger J. Griffin, Richard BaylissAbstract:Background Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein Kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-Related Kinase family that is essential for completion of the sexual development of the parasite.
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An Essential Role for the Plasmodium Nek-2 NIMA-Related Protein Kinase in the Sexual Development of Malaria Parasites*
2016Co-Authors: Oliver Billker, Christian DoerigAbstract:The molecular control of cell division and development in malaria parasites is far from understood.We previously showed that a Plasmodium gametocyte-specific NIMA-Related protein Kinase, nek-4, is required for completion of meiosis in the ooki-nete, the motile form that develops from the zygote in the mos-quito vector. Here, we show that another NIMA-Related Kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falcipa-rum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that dis-ruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete develop-ment. This has implications (i) in our understanding of sexua
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The Plasmodium falciparum, NIMA-Related Kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation.
Malaria journal, 2012Co-Authors: Luc Reininger, Andrew Tomlins, Sylke Müller, Christian Doerig, Miguel GarciaAbstract:Background Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite’s complex biology.
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The Plasmodium falciparum, NIMA-Related Kinase Pfnek-4: A marker for asexual parasites committed to sexual differentiation
Malaria Journal, 2012Co-Authors: Luc Reininger, Andrew Tomlins, Sylke Müller, Miguel Garcia, Christian DoerigAbstract:BACKGROUND: Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. METHODS: The expression and function of the NIMA-Related Kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. RESULTS: The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. CONCLUSIONS: The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation.
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an essential role for the plasmodium nek 2 NIMA Related protein Kinase in the sexual development of malaria parasites
Journal of Biological Chemistry, 2009Co-Authors: Luc Reininger, Christian Doerig, Oliver Billker, Rita Tewari, Clare Fennell, Zoe Holland, J Dean P Goldring, Lisa C RanfordcartwrightAbstract:The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-Related protein Kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-Related Kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission.
Vuk Stambolic - One of the best experts on this subject based on the ideXlab platform.
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NEK10 tyrosine phosphorylates p53 and controls its transcriptional activity
Oncogene, 2020Co-Authors: Nasir Haider, Previn Dutt, Vuk Stambolic, Bert Kooij, Luis Palomero, Miquel Angel Pujana, Michael YaffeAbstract:In response to genotoxic stress, multiple Kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NIMA-Related Kinase (NEK) family ( NEK1 , NEK10 , and NEK11 ) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10’s unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10 -deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53 -target gene expression is an independent predictor of a favorable outcome in breast cancers.
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nek family of Kinases in cell cycle checkpoint control and cancer
Cell Division, 2011Co-Authors: Previn Dutt, Larissa S Moniz, Nasir Haider, Vuk StambolicAbstract:Early studies in lower Eukaryotes have defined a role for the members of the NIMA Related Kinase (Nek) family of protein Kinases in cell cycle control. Expansion of the Nek family throughout evolution has been accompanied by their broader involvement in checkpoint regulation and cilia biology. Moreover, mutations of Nek family members have been identified as drivers behind the development of ciliopathies and cancer. Recent advances in studying the physiological roles of Nek family members utilizing mouse genetics and RNAi-mediated knockdown are revealing intricate associations of Nek family members with fundamental biological processes. Here, we aim to provide a comprehensive account of our understanding of Nek Kinase biology and their involvement in cell cycle, checkpoint control and cancer.
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nek10 mediates g2 m cell cycle arrest and mek autoactivation in response to uv irradiation
Molecular and Cellular Biology, 2011Co-Authors: Larissa S Moniz, Vuk StambolicAbstract:Appropriate cell cycle checkpoint control is essential for the maintenance of cell and organismal homeostasis. Members of the Nek (NIMA-Related Kinase) family of serine/threonine protein Kinases have been implicated in the regulation of various aspects of the cell cycle. We explored the cellular functions of Nek10, a novel member of the Nek family, and demonstrate a role for Nek10 in the cellular UV response. Nek10 was required for the activation of extracellular signal-regulated Kinase 1/2 (ERK1/2) signaling upon UV irradiation but not in response to mitogens, such as epidermal growth factor stimulation. Nek10 physically associated with Raf-1 and MEK1 in a Raf-1-dependent manner, and the formation of this complex was necessary for Nek10-mediated MEK1 activation. Nek10 did not affect the Kinase activity of Raf-1 but instead promoted the autophosphorylation-dependent activation of MEK1. The appropriate maintenance of the G(2)/M checkpoint following UV irradiation required Nek10 expression and ERK1/2 activation. Taken together, our results uncover a role for Nek10 in the cellular response to UV irradiation.
Xiaoping Chen - One of the best experts on this subject based on the ideXlab platform.
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micro ribonucleic acid 23a 3p prevents the onset of type 2 diabetes mellitus by suppressing the activation of nucleotide binding oligomerization like receptor family pyrin domain containing 3 inflammatory bodies caused pyroptosis through negatively r
Journal of Diabetes Investigation, 2021Co-Authors: Hongye Chang, Hongjuan Chang, Tuanjie Cheng, Garrick D. Lee, Xiaoping ChenAbstract:Aims/introduction Micro-ribonucleic acids (miRNAs) possess crucial functions in governing metabolisms associated with type 2 diabetes mellitus. This study aimed to investigate the role of miR-23a-3p in pyroptosis caused by nucleotide-binding oligomerization-like receptor family pyrin domain containing 3 (NLRP3) inflammatory body activation, thereby reducing the occurrence of type 2 diabetes mellitus. Materials and methods miR-23a-3p and NIMA-Related Kinase 7 (NEK7) expression in type 2 diabetes mellitus patients and rat models was examined. Dual-luciferase reporter gene experiments were used to verify the targeting relationship between miR-23a-3p and NEK7. Bone marrow-derived macrophages were transfected with miR-23a-3p mimic, miR-23a-3p inhibitor or short hairpin NEK7 and were treated with a specific activator of NLRP3 inflammatory body (lipopolysaccharide + adenosine-5'-triphosphate) to evaluate expression of NEK7, miR-23a-3p, gasdermin D p30, pro-caspase-1 and caspase-1 in cells, and interleukin-1β and tumor necrosis factor-α in supernatant. Type 2 diabetes mellitus rat models were used to observe the influences of miR-23a-3p, NEK7 and NLRP3 inflammatory body on pyroptosis and type 2 diabetes mellitus in vivo. Results NEK7 was overexpressed, whereas miR-23a-3p was underexpressed in patients and rat models with type 2 diabetes mellitus. NEK7 was a target gene of miR-23a-3p. After the addition of lipopolysaccharide + adenosine-5'-triphosphate in bone marrow-derived macrophages, the expression of miR-23a-3p subsequently declined. Furthermore, the addition of lipopolysaccharide + adenosine-5'-triphosphate elevated NEK7, NLRP3, pro-caspase-1, cle-caspase-1 and gasdermin D p30 expressions in bone marrow-derived macrophages, and enhanced levels of interleukin-1β and tumor necrosis factor-α in the supernatant, accompanied with conspicuous cell pyroptosis, which was reversed after miR-23a-3p overexpression and NEK7 silencing. miR-23a-3p overexpression alleviated liver and kidney damage in type 2 diabetes mellitus rats, and reduced NLRP3-induced pyroptosis. Conclusions Targeting NEK7 by miR-23a-3p could reduce NLRP3-induced pyroptosis, and assuage liver and kidney injuries in type 2 diabetes mellitus rats.
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Micro‐ribonucleic acid‐23a‐3p prevents the onset of type 2 diabetes mellitus by suppressing the activation of nucleotide‐binding oligomerization‐like receptor family pyrin domain containing 3 inflammatory bodies‐caused pyroptosis through negatively r
Journal of diabetes investigation, 2020Co-Authors: Hongye Chang, Hongjuan Chang, Tuanjie Cheng, Garrick D. Lee, Xiaoping ChenAbstract:Aims/introduction Micro-ribonucleic acids (miRNAs) possess crucial functions in governing metabolisms associated with type 2 diabetes mellitus. This study aimed to investigate the role of miR-23a-3p in pyroptosis caused by nucleotide-binding oligomerization-like receptor family pyrin domain containing 3 (NLRP3) inflammatory body activation, thereby reducing the occurrence of type 2 diabetes mellitus. Materials and methods miR-23a-3p and NIMA-Related Kinase 7 (NEK7) expression in type 2 diabetes mellitus patients and rat models was examined. Dual-luciferase reporter gene experiments were used to verify the targeting relationship between miR-23a-3p and NEK7. Bone marrow-derived macrophages were transfected with miR-23a-3p mimic, miR-23a-3p inhibitor or short hairpin NEK7 and were treated with a specific activator of NLRP3 inflammatory body (lipopolysaccharide + adenosine-5'-triphosphate) to evaluate expression of NEK7, miR-23a-3p, gasdermin D p30, pro-caspase-1 and caspase-1 in cells, and interleukin-1β and tumor necrosis factor-α in supernatant. Type 2 diabetes mellitus rat models were used to observe the influences of miR-23a-3p, NEK7 and NLRP3 inflammatory body on pyroptosis and type 2 diabetes mellitus in vivo. Results NEK7 was overexpressed, whereas miR-23a-3p was underexpressed in patients and rat models with type 2 diabetes mellitus. NEK7 was a target gene of miR-23a-3p. After the addition of lipopolysaccharide + adenosine-5'-triphosphate in bone marrow-derived macrophages, the expression of miR-23a-3p subsequently declined. Furthermore, the addition of lipopolysaccharide + adenosine-5'-triphosphate elevated NEK7, NLRP3, pro-caspase-1, cle-caspase-1 and gasdermin D p30 expressions in bone marrow-derived macrophages, and enhanced levels of interleukin-1β and tumor necrosis factor-α in the supernatant, accompanied with conspicuous cell pyroptosis, which was reversed after miR-23a-3p overexpression and NEK7 silencing. miR-23a-3p overexpression alleviated liver and kidney damage in type 2 diabetes mellitus rats, and reduced NLRP3-induced pyroptosis. Conclusions Targeting NEK7 by miR-23a-3p could reduce NLRP3-induced pyroptosis, and assuage liver and kidney injuries in type 2 diabetes mellitus rats.
Terje Johansen - One of the best experts on this subject based on the ideXlab platform.
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NIMA Related Kinase 9 mediated phosphorylation of the microtubule associated lc3b protein at thr 50 suppresses selective autophagy of p62 sequestosome 1
Journal of Biological Chemistry, 2020Co-Authors: Birendra Kumar Shrestha, Mads Skytte Rasmussen, Yakubu Princely Abudu, Jackansgar Bruun, Kenneth Bowitz Larsen, Endalkachew Ashenafi Alemu, Eva Sjottem, Trond Lamark, Terje JohansenAbstract:Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine Kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein Kinase Cζ (PKCζ) and never in mitosis A (NIMA)-Related Kinase 9 (NEK9). All three Kinases phosphorylated LC3B Thr-50 in vitro. A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.
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NIMA Related Kinase 9 mediated phosphorylation of the microtubule associated lc3b protein at thr 50 suppresses selective autophagy of p62 sequestosome 1
Journal of Biological Chemistry, 2020Co-Authors: Birendra Kumar Shrestha, Mads Skytte Rasmussen, Yakubu Princely Abudu, Jackansgar Bruun, Kenneth Bowitz Larsen, Endalkachew Ashenafi Alemu, Eva Sjottem, Trond Lamark, Terje JohansenAbstract:Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine Kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein Kinase Czeta (PKCzeta) and never in mitosis A (NIMA)-Related Kinase 9 (NEK9). All three Kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCzeta knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.
Jinbo Liu - One of the best experts on this subject based on the ideXlab platform.
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NIMA Related Kinase 7 amplifies nlrp3 inflammasome pro inflammatory signaling in microglia macrophages and mice models of spinal cord injury
Experimental Cell Research, 2021Co-Authors: Zhiwen Song, Shiwu Guo, Yuwei Chen, Honghai Wang, Jinlong Zhang, Jinbo LiuAbstract:Abstract Background NIMA-Related Kinase-7 (NEK7) is a serine/threonine Kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined. Methods In this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP). Results Here, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord. Conclusion NEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.
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NIMA-Related Kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury.
Experimental cell research, 2020Co-Authors: Zhiwen Song, Shiwu Guo, Yuwei Chen, Honghai Wang, Jinlong Zhang, Jinbo LiuAbstract:Abstract Background NIMA-Related Kinase-7 (NEK7) is a serine/threonine Kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined. Methods In this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP). Results Here, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord. Conclusion NEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.