scholarly journals Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death

2016 ◽  
Vol 36 (1) ◽  
Author(s):  
Katja Hrovat Arnež ◽  
Michaela Kindlova ◽  
Nilesh J. Bokil ◽  
James M. Murphy ◽  
Matthew J. Sweet ◽  
...  
Keyword(s):  
Cell Death ◽  
Human Monocyte ◽  
Kinase Domain ◽  
Terminal Region ◽  
Activation Loop ◽  
Phosphorylation Sites ◽  
Mixed Lineage Kinase

We show that mixed lineage kinase domain-like (MLKL) isoform 2, which lacks the pseudokinase domain and activation loop phosphorylation sites, is a more potent activator of cell death compared with MLKL isoform 1. Both MLKL isoforms are expressed in human monocyte-derived macrophages.

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

scholarly journals Diverse Sequence Determinants Control Human and Mouse Receptor Interacting Protein 3 (RIP3) and Mixed Lineage Kinase domain-Like (MLKL) Interaction in Necroptotic Signaling

2013 ◽  
Vol 288 (23) ◽  
pp. 16247-16261 ◽  
Author(s):  
Wanze Chen ◽  
Zhenru Zhou ◽  
Lisheng Li ◽  
Chuan-Qi Zhong ◽  
Xinru Zheng ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Flanking Sequence ◽  
Interacting Protein ◽  
Mixed Lineage Kinase ◽  
Signaling Complex ◽  
Evolutionarily Conserved ◽  
Mouse Cells ◽  
Human And Mouse

Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.

scholarly journals Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

Cell Research ◽  
2013 ◽  
Vol 24 (1) ◽  
pp. 105-121 ◽  
Author(s):  
Xin Chen ◽  
Wenjuan Li ◽  
Junming Ren ◽  
Deli Huang ◽  
Wan-ting He ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Kinase Domain ◽  
Necrotic Cell Death ◽  
Necrotic Cell ◽  
Mixed Lineage Kinase

scholarly journals Elevated Serum Levels of Mixed Lineage Kinase Domain-Like Protein Predict Survival of Patients during Intensive Care Unit Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Mihael Vucur ◽  
Christoph Roderburg ◽  
Lukas Kaiser ◽  
Anne Theres Schneider ◽  
Sanchari Roy ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Cell Death ◽  
Intensive Care ◽  
Critical Illness ◽  
Critically Ill Patients ◽  
Serum Levels ◽  
Kinase Domain ◽  
Healthy Controls ◽  
Mixed Lineage Kinase ◽  
Icu Treatment

Mixed lineage kinase domain-like (MLKL), a crucial regulator of necroptotic cell death, was shown to play a role in inflammatory diseases. However, its role as a biomarker in critical illness and sepsis is currently unknown. We analyzed serum levels of MLKL in 136 critically ill patients at admission to the intensive care unit (ICU) and after three days of ICU treatment. Results were compared with 36 healthy controls and correlated with clinical and laboratory patients’ data. MLKL serum levels of critically ill patients at admission to the ICU were similar compared to healthy controls. At ICU admission, MLKL serum concentrations were independent of disease severity, presence of sepsis, and etiology of critical illness. In contrast, median serum levels of MLKL after three days of ICU treatment were significantly lower compared to those at admission to the ICU. While serum levels of MLKL at admission were not predictive for short-term survival during ICU treatment, elevated MLKL concentrations at day three were an independent negative predictor of patients’ ICU survival. Thus, elevated MLKL levels after three days of ICU treatment were predictive for patients’ mortality, indicating that sustained deregulated cell death is associated with an adverse prognosis in critical illness.

scholarly journals Plant mixed lineage kinase domain-like proteins limit biotrophic pathogen growth

2019 ◽  
Author(s):  
Lisa Mahdi ◽  
Menghang Huang ◽  
Xiaoxiao Zhang ◽  
Ryohei Thomas Nakano ◽  
Leïla Brulé Kopp ◽  
...  
Keyword(s):  
Cell Death ◽  
Host Cell ◽  
Kinase Domain ◽  
Protein Family ◽  
Mixed Lineage Kinase ◽  
Helical Bundles ◽  
Cryo Electron Microscopy ◽  
Host Cell Death ◽  
A Cell ◽  
Immunity Mechanism

AbstractMixed lineage kinase domain-like (MLKL) protein mediates necroptotic cell death in vertebrates. We report here the discovery of a conserved protein family across seed plants that is structurally homologous to vertebrate MLKL. TheArabidopsis thalianagenome encodes three MLKLs with overlapping functions in limiting growth of obligate biotrophic fungal and oomycete pathogens. Although displaying a cell death activity mediated by N-terminal helical bundles, termed HeLo domain,AtMLKL-dependent immunity can be separated from host cell death. Cryo-electron microscopy structures ofAtMLKLs reveal a tetrameric configuration, in which the pseudokinase domain and brace region bury the HeLo-domains, indicative of an auto-repressed complex. We also show the association of twoAtMLKLs with microtubules. These findings, coupled with resistance-enhancing activity and altered microtubule association of a phosphomimetic mutation in the pseudokinase domain ofAtMLKL1, point to a cell death-independent immunity mechanism.One Sentence SummaryPlants have a protein family that is structurally homologous to vertebrate mixed lineage kinase domain-like protein, which induces necroptotic cell death, but these plant proteins can confer immunity without host cell death.

scholarly journals Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

2022 ◽  
Author(s):  
Seongmin Yoon ◽  
Konstantin Bogdanov ◽  
David Wallach
Keyword(s):  
Cell Death ◽  
Defense Mechanisms ◽  
Kinase Domain ◽  
Intracellular Bacteria ◽  
Endosomal Trafficking ◽  
Polyubiquitin Chains ◽  
Mixed Lineage Kinase ◽  
Lysine Residues ◽  
Covalent Modifications ◽  
Bacterial Yield

AbstractPhosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.

scholarly journals Oligomerization-driven MLKL ubiquitylation antagonises necroptosis

2021 ◽  
Author(s):  
Zikou Liu ◽  
Laura Francesca Dagley ◽  
Kristy Lynn Shield-Artin ◽  
Samuel Nicholas Young ◽  
Aleksandra Bankovacki ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Membrane Disruption ◽  
Fusion Strategy ◽  
Mixed Lineage Kinase ◽  
Independent Form ◽  
Lysine Residues ◽  
Kinetic Regulation

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome- dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licenses MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

scholarly journals RIPK3-Dependent Necroptosis Is Induced and Restricts Viral Replication in Human Astrocytes Infected With Zika Virus

2021 ◽  
Vol 11 ◽  
Author(s):  
Chunxia Wen ◽  
Yufeng Yu ◽  
Chengfeng Gao ◽  
Xian Qi ◽  
Carol J. Cardona ◽  
...  
Keyword(s):  
Cell Death ◽  
Viral Replication ◽  
Zika Virus ◽  
Developmental Disorders ◽  
Viral Disease ◽  
Kinase Domain ◽  
Zikv Infection ◽  
Mixed Lineage Kinase ◽  
Disease Outcomes ◽  
Human Astrocytes

Apoptosis, pyroptosis and necroptosis are regulated processes of cell death which can be crucial for viral disease outcomes in hosts because of their effects on viral pathogenicity and host resistance. Zika virus (ZIKV) is a mosquito-borne flavivirus, which infects humans and can cause neurological disorders. Neural developmental disorders and microcephaly could occur in infected fetuses. Several types of nervous cells have been reported to be susceptible to ZIKV infection. Human astrocytes play important roles in the nutritional support and defense of neurons. In this study, we show that human astrocytes are susceptible to ZIKV infection and undergo progressive cell death after infection. In infected astrocytes we detected no cleavage or activation of pro-caspase-3 and pro-caspase-1. Apoptotic substrates and increased secretion of interleukin (IL)-1β or IL-18 were not detected, either. These ruled out the occurrence of apoptosis or pyroptosis in ZIKV-infected astrocytes. We detected, however, an increase of phosphorylated receptor-interacting serine/threonine-protein kinase (RIPK)1, RIPK3, and mixed lineage kinase domain-like (MLKL) protein, indicating that programmed necrosis, or necroptosis, was induced in infected astrocytes. The phosphorylation and cell death were inhibited in cells pre-treated with GSK’872, an inhibitor of RIPK3, while inhibition of RIPK1 with an inhibitor, Necrostatin-1, had no effect, suggesting that ZIKV-induced necroptosis was RIPK1-independent in astrocytes. Consistent with this finding, the inhibition of RIPK1 had no effect on the phosphorylation of MLKL. We showed evidence that MLKL phosphorylation was RIPK3-dependent and ZBP-1, which could stimulate RIPK3, was upregulated in ZIKV-infected astrocytes. Finally, we demonstrated that in GSK’872-pre-treated astrocytes, viral replication increased significantly, which indicates that necroptosis may be protective against viral replication in astrocytes. Our finding that astrocytes uniquely underwent necroptosis in response to ZIKV infection provides insight and helps us better understand the viral pathogenesis in the ZIKV-infected central nervous system.

scholarly journals The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation

1998 ◽  
Vol 333 (3) ◽  
pp. 631-636 ◽  
Author(s):  
Pilar GARCIA-PARAMIO ◽  
Yolanda CABRERIZO ◽  
Frederic BORNANCIN ◽  
Peter J. PARKER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Activation Loop ◽  
Phosphorylation Sites ◽  
Inhibitory Protein ◽  
Kinase C ◽  
Broad Specificity

Dominant negative properties are conferred on protein kinase (PK) Cα by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCζ mutants (atypical isotype) inhibited both PKCα (classical isotype) and PKCε (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCα, consistent with the expected mode of action. In the case of the PKCα mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.

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