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Biochimica Et Biophysica Acta.... Jun 2018Human α-adrenoceptors (α-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of...
Human α-adrenoceptors (α-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/β, phosphorylate α-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.
Topics: HEK293 Cells; Humans; MAP Kinase Signaling System; Phosphorylation; Protein Domains; Protein Structure, Secondary; Receptors, Adrenergic, alpha-1
PubMed: 29551601
DOI: 10.1016/j.bbamcr.2018.03.006 -
International Journal of Molecular... Nov 2019Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination...
BACKGROUND
Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination of the Beclin-1 protein complex. Rubicon is a Beclin-1 associated protein that suppresses autophagy by impairing the activity of the class III PI3K, Vps34. However, very little is known about the molecular mechanisms that regulate Rubicon function.
METHODS
In this study, co-immunoprecipitation and kinase assays were used to investigate the ability of Hormonally Upregulated Neu-associated Kinase (HUNK) to bind to and phosphorylate Rubicon. LC3B was monitored by immunofluorescence and immunoblotting to determine whether phosphorylation of Rubicon by HUNK controls the autophagy suppressive function of Rubicon.
RESULTS
Findings from this study identify Rubicon as a novel substrate of HUNK and show that phosphorylation of Rubicon inhibits its function, promoting autophagy.
Topics: Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Cells, Cultured; HEK293 Cells; Humans; Phagosomes; Phosphorylation; Protein Serine-Threonine Kinases
PubMed: 31752345
DOI: 10.3390/ijms20225813 -
Autophagy Feb 2022ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome...
ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale kinase assay and characterized 187 phosphosites on 157 proteins as ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.
Topics: Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Intracellular Signaling Peptides and Proteins; Mice; Phosphorylation; Protein Phosphatase 2
PubMed: 34839766
DOI: 10.1080/15548627.2021.2002546 -
Progress in Biophysics and Molecular... 2009The activity of a protein can be reversibly modulated by post-translational, covalent modifications, such as phosphorylation and dephosphorylation. In many cases, the... (Review)
Review
The activity of a protein can be reversibly modulated by post-translational, covalent modifications, such as phosphorylation and dephosphorylation. In many cases, the modulated protein may be phosphorylated by the same kinase on many different amino acid residues. Such multisite phosphorylations may occur progressively (during a single binding event of kinase to substrate) or distributively (the kinase dissociates from its substrate after each phosphorylation reaction). If a protein is phosphorylated by a distributive multisite mechanism, then the net activity of a population of these protein molecules can be a highly nonlinear function of the ratio of activities of the kinase and phosphatase enzymes. If the multiply phosphorylated protein is embedded in a positive feedback loop with its kinase and/or phosphatase, then the network may exhibit robust bistable behavior. Using numerical simulations and bifurcation theory, we study the properties of a particular bistable reaction network motivated by the antagonistic relationship between cyclin-dependent kinase and its multiply phosphorylated target, Cdh1, which is involved in the degradation of cyclin molecules. We characterize the bistable switch in terms of (i) the mechanism of distributive phosphorylation (ordered or disordered), (ii) the number of phosphorylation sites on the target protein, (iii) the effect of phosphorylation on the target protein (abrupt or progressive inactivation), and (iv) the effects of stochastic fluctuations in small cells with limited numbers of kinase, phosphatase and target proteins.
Topics: Animals; Feedback, Physiological; Models, Biological; Phosphorylation; Proteins; Stochastic Processes
PubMed: 19523976
DOI: 10.1016/j.pbiomolbio.2009.06.004 -
Biochimica Et Biophysica Acta.... Jan 2024AP2 forms AP2 CCV with clathrin and over 60 additional coat proteins. Due to this complexity, we have a limited understanding of CCV life cycle regulation. Synapses...
AP2 forms AP2 CCV with clathrin and over 60 additional coat proteins. Due to this complexity, we have a limited understanding of CCV life cycle regulation. Synapses contain canonical AP2 CCV, canCCV, and more stable, thereby longer lived, AP2 CCV. The more stable AP2 CCV can be distinguished from canCCV due to the stable binding of Hsc70 to clathrin. The AP1/σ1B complex knockout leads to impaired synaptic vesicle recycling and altered endosomal protein sorting. This causes as a secondary phenotype the twofold upregulation of endocytosis by canCCV and by more stable AP2 CCV. These stable CCV are more stabilized than their wt counterpart, hence stCCV. They have less of the uncoating proteins synaptojanin1 and Hsc70, and more of the coat stabilizing AAK1. Hsc70 clathrin dissociation activity is regulated by complex phosphorylation patterns. Two major groups of hyper- and of hypo-phosphorylated Hsc70 proteins are formed. The latter are enriched in wt stable CCV and stabilized stCCV. Hsc70 T265 phosphorylation regulates binding of CaM/Ca. CaM/Ca binding to the T265 domain blocks Hsc70 homodimerization and its concentration in stCCV required for clathrin disassembly. Kinases DYRK1A and CaMK-IIδ can phosphorylate T265 preventing CaM/Ca binding. Their and the levels of STK38L and STK39/Cab39, which are able to phosphorylate additional Hsc70 residues are reduced in stCCV. The stCCV pathway sorts specifically the cell adhesion proteins CHL1 and Neurocan, supporting our model of that the stCCV pathway fulfills specific functions in synaptic plasticity.
Topics: Phosphorylation; Calmodulin; Longevity; Cell Adhesion; Clathrin; Cell Adhesion Molecules; Protein Transport
PubMed: 37926156
DOI: 10.1016/j.bbamcr.2023.119611 -
Cancer Science Mar 2006Intermediate filaments (IF) form the structural framework of the cytoskeleton. Although histopathological detection of IF proteins is utilized for examining cancer... (Review)
Review
Intermediate filaments (IF) form the structural framework of the cytoskeleton. Although histopathological detection of IF proteins is utilized for examining cancer specimens as reliable markers, the molecular mechanisms by which IF are involved in the biology of cancer cells are still unclear. We found that site-specific phosphorylation of IF proteins induces the disassembly of filament structures. To further dissect the in vivo spatiotemporal dynamics of IF phosphorylation, we developed site- and phosphorylation state-specific antibodies. Using these antibodies, we detected kinase activities that specifically phosphorylate type III IF, including vimentin, glial fibrillary acidic protein and desmin, during mitosis. Cdk1 phosphorylates vimentin-Ser55 from prometaphase to metaphase, leading to the recruitment of Polo-like kinase 1 (Plk1) to vimentin. Upon binding to Phospho-Ser55 of vimentin, Plk1 is activated, and then phosphorylates vimentin-Ser82. During cytokinesis, Rho-kinase and Aurora-B specifically phosphorylate IF at the cleavage furrow. IF phosphorylation by Cdk1, Plk1, Rho-kinase and Aurora-B plays an important role in the local IF breakdown, and is essential for the efficient segregation of IF networks into daughter cells. As another part of our research on IF, we have set out to find the binding partners with simple epithelial keratin 8/18. We identified tumor necrosis factor receptor type 1-associated death domain protein (TRADD) as a keratin 18-binding protein. Together with data from other laboratories, it is proposed that simple epithelial keratins may play a role in modulating the response to some apoptotic signals. Elucidation of the precise molecular functions of IF is expected to improve our understanding of tumor development, invasion and metastasis.
Topics: Animals; Antibodies; Cell Transformation, Neoplastic; Humans; Intermediate Filaments; Keratins; Mitosis; Phosphorylation
PubMed: 16542212
DOI: 10.1111/j.1349-7006.2006.00161.x -
Protein Science : a Publication of the... Mar 2013Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that...
Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that proteins (galectin-1, ubiquitin, RNase, cytochrome c, myoglobin, and lysozyme) under reducing conditions with dithiothreitol (DTT) become alkylated at lysine-N(ζ) groups and O-phosphorylated at serine and threonine residues. These adduction reactions only occur in the presence of monophosphate, potassium, trace metals Fe/Cu, and oxygen, and are promoted by reactive oxygen species (ROS) generated via DTT oxidation. Superoxide mediates the chemistry, because superoxide dismutase inhibits the reaction, and hydroxyl and phosphoryl radicals are also likely involved. While lysine alkylation accounts for most of the adduction, low levels of phosphorylation are also observed at some serine and threonine residues, as determined by western blotting and MS fingerprinting. The adducted alkyl group is found to be a fragment of DTT that forms a Schiff base at lysine N(ζ) groups. Although its exact chemical structure remains unknown, the DTT fragment includes a SH group and a --CHOH--CH₂-- group. Chemical adduction appears to be promoted in the context of a well-folded protein, because some adducted sites in the proteins studied are considerably more reactive than others and the reaction occurs to a lesser extent with shorter, unfolded peptides and not at all with small organic molecules. A structural signature involving clusters of positively charged and other polar groups appears to facilitate the reaction. Overall, our findings demonstrate a novel reaction for DTT-mediated ROS chemistry with proteins.
Topics: Alkylation; Animals; Catalysis; Dithiothreitol; Humans; Lysine; Mutant Proteins; Oxidation-Reduction; Phosphorylation; Protein Conformation; Protein Folding; Proteins; Reactive Oxygen Species; Reducing Agents; Schiff Bases; Serine; Threonine
PubMed: 23315912
DOI: 10.1002/pro.2214 -
The Journal of Biological Chemistry Aug 2005Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation...
Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.
Topics: Amino Acid Sequence; Animals; Cattle; Cell Line; Consensus Sequence; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Darkness; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; G-Protein-Coupled Receptor Kinases; Humans; Kidney; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Phosphoserine; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Proteins; Rhodopsin; Rod Cell Outer Segment; Urea
PubMed: 15946941
DOI: 10.1074/jbc.M505117200 -
The Journal of Biological Chemistry Apr 2016P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes...
P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was ∼1 μmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 μmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.
Topics: ATP-Binding Cassette Transporters; Calcium; Endoplasmic Reticulum Stress; Phosphorylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26858246
DOI: 10.1074/jbc.M115.695122 -
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