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The EMBO Journal Jun 2022PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3...
PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
Topics: Phosphorylation; Protein Domains; Protein Kinases; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 35491809
DOI: 10.15252/embj.2021109460 -
Journal of Lipid Research Mar 2024Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To...
Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.
Topics: Animals; Mice; Phosphorylation; Diglycerides; Diacylglycerol Kinase
PubMed: 38272356
DOI: 10.1016/j.jlr.2024.100506 -
Journal of Neurochemistry Jul 2010Brain aromatic L-amino acid decarboxylase (AAAD) is subject to regulation, and phosphorylation might be involved in the short-term activation of the enzyme. Sites for...
Brain aromatic L-amino acid decarboxylase (AAAD) is subject to regulation, and phosphorylation might be involved in the short-term activation of the enzyme. Sites for serine/threonine phosphorylation are present in the deduced amino acid sequence of AAAD, and cAMP-dependent protein kinase phosphorylates and activates neuronal AAAD in vitro. We now report that cGMP-dependent protein kinase (PKG) is able to phosphorylate and activate neuronal AAAD. In an in vitro kinase assay, immunoprecipitated native and recombinant mouse brain AAAD was rapidly phosphorylated by exogenous PKGIalpha. When added to striatal homogenates, PKGIalpha increased AAAD activity in a temporal fashion similar to phosphorylation. Recombinant AAAD was also activated by the kinase demonstrating a direct effect. Native enzyme activation was moderate and characterized by increased V(max) and K(m) for L-DOPA. A PKG peptide inhibitor prevented AAAD phosphorylation and activation providing specificity, and causally linking the two events. Together, the findings provide evidence for PKGIalpha-dependent phosphorylation and activation of neuronal AAAD in vitro, and introduce AAAD as a putative PKGIalpha substrate. Neuronal AAAD is best known for its role in the biosynthesis of catecholamines, indoleamines and trace amines in the nervous system, and the biological importance of PKGIalpha phosphorylation in these processes remains to be determined.
Topics: Animals; Aromatic-L-Amino-Acid Decarboxylases; Brain; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; In Vitro Techniques; Male; Mice; Neurons; Phosphorylation
PubMed: 20456015
DOI: 10.1111/j.1471-4159.2010.06784.x -
The Plant Cell Jul 2021Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is...
Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.
Topics: Abscisic Acid; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Oryza; Oxidative Stress; Phosphorylation; Phosphothreonine; Plant Proteins; Protein Binding; Signal Transduction; Stress, Physiological; Water
PubMed: 33630095
DOI: 10.1093/plcell/koab071 -
The Journal of Biological Chemistry Jun 2002Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it...
Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).
Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cytoskeletal Proteins; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Mutation; Phosphorylation; Structure-Activity Relationship; Trans-Activators; beta Catenin
PubMed: 11967263
DOI: 10.1074/jbc.M201364200 -
Current Protocols Sep 2022Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that...
Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).
Topics: Adenosine Triphosphate; Catalysis; Immunoprecipitation; Phosphoproteins; Phosphorylation
PubMed: 36135312
DOI: 10.1002/cpz1.539 -
International Journal of Molecular... Nov 2023Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in...
Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in various cancer types, with tyrosine phosphorylation shown to play a critical role in driving cancer cell migration. However, the specific impact of the distinct tyrosine phosphorylation events of paxillin in the progression of breast cancer remains to be fully elucidated. Here, we found that paxillin overexpression in breast cancer tissue is associated with a patient's poor prognosis. Paxillin knockdown inhibited the migration and invasion of breast cancer cells. Furthermore, the phosphorylation of paxillin tyrosine residue 31 (Tyr31) was significantly increased upon the TGF-β1-induced migration and invasion of breast cancer cells. Inhibiting Fyn activity or silencing Fyn decreases paxillin Tyr31 phosphorylation. The wild-type and constitutively active Fyn directly phosphorylate paxillin Tyr31 in an in vitro system, indicating that Fyn directly phosphorylates paxillin Tyr31. Additionally, the non-phosphorylatable mutant of paxillin at Tyr31 reduces actin stress fiber formation, migration, and invasion of breast cancer cells. Taken together, our results provide direct evidence that Fyn-mediated paxillin Tyr31 phosphorylation is required for breast cancer migration and invasion, suggesting that targeting paxillin Tyr31 phosphorylation could be a potential therapeutic strategy for mitigating breast cancer metastasis.
Topics: Female; Humans; Breast Neoplasms; Cell Movement; Paxillin; Phosphorylation; Tyrosine
PubMed: 37958964
DOI: 10.3390/ijms242115980 -
Biochimica Et Biophysica Acta. Gene... Nov 2022The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of...
The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.
Topics: Chromatin; Phosphorylation; Histones; DNA Damage; DNA
PubMed: 36280132
DOI: 10.1016/j.bbagrm.2022.194887 -
Cellular and Molecular Life Sciences :... Jan 2020Transforming growth factor (TGF)-β signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological... (Review)
Review
Transforming growth factor (TGF)-β signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-β signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-β receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-β can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-β signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-β signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.
Topics: Animals; Gene Expression; Humans; Phosphorylation; Signal Transduction; Smad Proteins; Transforming Growth Factor beta
PubMed: 31407020
DOI: 10.1007/s00018-019-03266-3 -
Biochimica Et Biophysica Acta. General... Nov 2022FcγRIIIa-FcεRIγ complexes, upon stimulation by antibodies, cluster to initiate intracellular signaling and activate natural killer (NK) cells. Intracellular signaling...
FcγRIIIa-FcεRIγ complexes, upon stimulation by antibodies, cluster to initiate intracellular signaling and activate natural killer (NK) cells. Intracellular signaling involves Lck phosphorylation of ITAMs of each monomer of a FcεRIγ homodimer in a FcγRIIIa-FcεRIγ complex and subsequent binding of two phosphotyrosines (pY) in tandem by a Syk family kinase. However, how FcR clustering triggers ITAM signaling is not resolved. Molecular modeling and dynamics (MD) simulations are applied to generate ensembles of structures of the FcγRIIIa and FcεRIγ homodimeric cytoplasmic tails of FcγRIIIa-FcεRIγ complexes based on the transmembrane helices and cytoplasmic tails spaced 120, 80, and 50 Å apart to model different extents of clustering. Site-identification by ligand competitive saturation method with Monte Carlo sampling (SILCS-MC) is used to model how Lck could phosphorylate a diversity of ITAM conformations. At 80 Å separation between FcγRIIIa-FcεRIγ complexes, Lck can perform multiple phosphorylations on individual and multiple ITAMs across complexes, including potential sequential phosphorylation events. Syk may then potentially bind the two pYs within a single ITAM in tandem in isolated FcγRIIIa-FcεRIγ complexes, as observed in CD3ε and ζ chains of T cell receptors by the Syk family kinase ZAP-70. In addition, at 50 Å separation between complexes, unique to natural killer cells over T cells, Syk could potentially bind in tandem to pYs in different ITAMs across FcγRIIIa-FcεRIγ complexes. Thus, we predict that an ensemble of spatial orientations of the ITAMS of FcγRIIIa-FcεRIγ complexes that occur upon clustering lead to ITAM phosphorylation by Lck and subsequent Syk activity thereby facilitating downstream signaling.
Topics: Killer Cells, Natural; Phosphorylation; Receptors, Antigen, T-Cell; Signal Transduction; Syk Kinase
PubMed: 35933027
DOI: 10.1016/j.bbagen.2022.130221