-
International Journal of Molecular... Feb 2023Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate...
Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.
Topics: Humans; Adenocarcinoma of Lung; Phosphorylation; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase
PubMed: 36769342
DOI: 10.3390/ijms24033023 -
Cancer Science Feb 2019Smad3, a major transcription factor in transforming growth factor-β (TGF-β) signaling, plays critical roles in both tumor-suppressive and pro-oncogenic functions. Upon... (Review)
Review
Smad3, a major transcription factor in transforming growth factor-β (TGF-β) signaling, plays critical roles in both tumor-suppressive and pro-oncogenic functions. Upon TGF-β stimulation, the C-terminal tail of Smad3 undergoes phosphorylation that is essential for canonical TGF-β signaling. The Smad3 linker region contains serine/threonine phosphorylation sites and can be phosphorylated by intracellular kinases, such as the MAPK family, cyclin-dependent kinase (CDK) family and glycogen synthase kinase-3β (GSK-3β). Previous reports based on cell culture studies by us and others showed that mutation of Smad3 linker phosphorylation sites dramatically intensifies TGF-β responses as well as growth-inhibitory function and epithelial-mesenchymal transition (EMT), suggesting that Smad3 linker phosphorylation suppresses TGF-β transcriptional activities. However, recent discoveries of Smad3-interacting molecules that preferentially bind phosphorylated Smad3 linker serine/threonine residues have shown a multitude of signal transductions that either enhance or suppress TGF-β responses associated with Smad3 turnover or cancer progression. This review aims at providing new insight into the perplexing mechanisms of TGF-β signaling affected by Smad3 linker phosphorylation and further attempts to gain insight into elimination and protection of TGF-β-mediated oncogenic and growth-suppressive signals, respectively.
Topics: Animals; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Neoplasms; Phosphorylation; Smad3 Protein; Transforming Growth Factor beta
PubMed: 30589983
DOI: 10.1111/cas.13922 -
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 -
Nature Communications May 2024Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites...
Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites on proteins and thereby regulates their function. Inspired by such machinery, regulating supramolecular functions using energy stored in reactive molecules has gained traction. Enzyme-free, synthetic systems that use dynamic phosphorylation to regulate supramolecular processes have not yet been reported, to our knowledge. Here, we show an enzyme-free reaction cycle that consumes the phosphorylating agent monoamidophosphate by transiently phosphorylating histidine and histidine-containing peptides. The phosphorylated species are labile and deactivate through hydrolysis. The cycle exhibits versatility and tunability, allowing for the dynamic phosphorylation of multiple precursors with a tunable half-life. Notably, we show the resulting phosphorylated products can regulate the peptide's phase separation, leading to active droplets that require the continuous conversion of fuel to sustain. The reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.
Topics: Phosphorylation; Peptides; Histidine; Adenosine Triphosphate; Hydrolysis
PubMed: 38760374
DOI: 10.1038/s41467-024-48571-z -
Journal of Proteomics Feb 2018Exposure of Wehi-231 B-cells to Hg for 5min resulted in concentration dependent changes in protein phosphorylations. Phosphorylation was quantified using mass...
UNLABELLED
Exposure of Wehi-231 B-cells to Hg for 5min resulted in concentration dependent changes in protein phosphorylations. Phosphorylation was quantified using mass spectrometry to analyze TiO and anti-pTyr antibody selected phosphopeptides from Wehi-231 digests. The most frequent and largest amplitude responses to Hg exposure were increased phosphorylation although a decrease was observed for 1% of phosphoproteins detected in the untreated cells. A subset of proteins responded with an increase in phosphorylation to Hg exposure at low micromolar concentrations. The majority of proteins required Hg over 20μM in order to increase phosphorylation. Ser/Thr phosphorylations are prominent in the cytoskeletal organization and the GTPase signaling systems and these systems are notable as the primary ones responding to the lowest concentrations of Hg. Systems that required higher concentrations of Hg to increase phosphorylation included immune receptor signaling. The proteins for which an increase in phosphorylation occurred at Hg above 20μM have a higher proportion of pTyr sites. Anti Ig stimulation of Wehi-231 cells confirmed that cytoskeletal protein phosphorylation and GTPase signaling are modulated in physiologically relevant B-cell receptor activation. Candidate kinases that respond to Hg exposure at the low μM concentrations include MAP Kinase 1, CaM Kinase II delta and PAK2.
SIGNIFICANCE
Mercury (Hg) is a wide spread environmental toxicant. Epidemiological and laboratory studies suggest that exposure to environmental Hg at current levels, which have been perceived to be non-toxic, may contribute to immune system dysfunction and autoimmune disease in humans and animals respectively. While we have previously shown that exposure of B lymphocytes to low levels of mercury interferes with B-cell receptor signaling mediated by post transcriptional phosphorylation events, overall the mechanism that is responsible for increased autoimmunity in mercury exposed human or animal populations is not well understood. The current study evaluated the dose dependent actions of mercury to change phosphorylation in the Wehi-231 cell line, an immature B-cell model in which actions of mercury on development of cell function can be evaluated. The study identified the cytoskeletal proteins as the most sensitive to modulation by mercury with changes in Ser/Thr phosphorylation being observed at the lowest concentrations of mercury. These findings indicate that the actions of mercury on B-cell immune function and development are at least in part likely mediated through changes in cytoskeletal protein phosphorylation.
Topics: Animals; B-Lymphocytes; Cell Line; Cytoskeleton; Dose-Response Relationship, Drug; Environmental Exposure; GTP Phosphohydrolases; Humans; Mass Spectrometry; Mercury; Phosphoproteins; Phosphorylation; Proteome; Serine; Signal Transduction; Threonine
PubMed: 29199152
DOI: 10.1016/j.jprot.2017.11.026 -
Cells Jul 2021CK2α/CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. Considering the role of CSNK2A1 in cancer progression and the...
CK2α/CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. Considering the role of CSNK2A1 in cancer progression and the phosphorylation of SIRT6 and the role of SIRT6 in chemoresistance through the DNA damage repair pathway, CSNK2A1 and SIRT6 might be involved in resistance to conventional anti-cancer therapies. We evaluated the expression of CSNK2A1 and phosphorylated SIRT6 in the 37 osteosarcoma patients and investigated the effects of CSNK2A1 and the phosphorylation of SIRT6 on Ser338 on resistance to the anti-cancer effects of doxorubicin. Higher expression of CSNK2A1 and phosphorylated SIRT6 was associated with shorter survival in osteosarcoma patients. U2OS and KHOS/NP osteosarcoma cells with induced overexpression of CSNK2A1 were resistant to the cytotoxic effects of doxorubicin, and the knock-down of CSNK2A1 potentiated the cytotoxic effects of doxorubicin. CSNK2A1 overexpression-mediated resistance to doxorubicin was associated with SIRT6 phosphorylation and the induction of the DNA damage repair pathway molecules. CSNK2A1- and SIRT6-mediated resistance to doxorubicin in vivo was attenuated via mutation of SIRT6 at the Ser338 phosphorylation site. Emodin, a CSNK2A1 inhibitor, potentiated the cytotoxic effects of doxorubicin in osteosarcoma cells. This study suggests that blocking the CSNK2A1-SIRT6-DNA damage repair pathway might be a new therapeutic stratagem for osteosarcomas.
Topics: Adult; Apoptosis; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; DNA Damage; DNA Repair; Doxorubicin; Drug Resistance, Neoplasm; Emodin; Female; Humans; Kaplan-Meier Estimate; Male; Multivariate Analysis; Phosphorylation; Prognosis; Proportional Hazards Models; Regression Analysis; Sirtuins
PubMed: 34359939
DOI: 10.3390/cells10071770 -
Journal of Neurochemistry Jan 2020Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of...
Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of catecholamine synthesis, is regulated by multiple neuronal signaling pathways through phosphorylation at serine 19 (Ser19), serine 31 (Ser31), and serine 40 (Ser40) located in the flexible, far N-terminal region of the regulatory domain. Phosphorylated Ser19 (pSer19) provides a binding site for 14-3-3 proteins, a family of multi-target binding adaptor proteins. We hypothesized that pSer19 and 14-3-3 binding can regulate access to the Ser31 and Ser40 sites and modulate the dynamics of their phosphorylation state. To avoid complications from upstream signal interactions and have good control of TH-phosphorylation and 14-3-3 binding stoichiometry, we used purified recombinant human TH and 14-3-3 dimer types. We found that pSer19 strongly stimulated Ser31 phosphorylation (4.6-fold), but inhibited pSer31 dephosphorylation (3.4-fold). Binding of 14-3-3ζ counteracted the stimulatory effect of pSer19 on phosphorylation at Ser31, but amplified the effect on its dephosphorylation. In contrast, phosphorylation at Ser19 had moderate effect on pSer40 dephosphorylation, but 14-3-3ζ binding inhibited dephosphorylation, an effect that was consistent across different homo- and heterodimeric 14-3-3s. Additional phosphorylation of Ser31 or Ser40 had little impact on the binding affinity of pSer19 TH to 14-3-3s. Mathematical modeling was performed to elucidate possible physiological implications of these observations. We propose a role of Ser19 and 14-3-3 proteins as modulators of TH phosphorylation in response to neuronal co-signaling events. These mechanisms add to our understanding of the multifaceted roles of phosphorylation and adaptor proteins in cellular signaling.
Topics: 14-3-3 Proteins; Animals; Humans; Models, Theoretical; PC12 Cells; Phosphorylation; Rats; Recombinant Proteins; Serine; Tyrosine 3-Monooxygenase
PubMed: 31529487
DOI: 10.1111/jnc.14872 -
Science Signaling Apr 2021The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether...
The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.
Topics: Amino Acid Motifs; Animals; Mechanistic Target of Rapamycin Complex 2; Mice; Peptides; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-akt
PubMed: 33850054
DOI: 10.1126/scisignal.abe4509 -
Journal of Proteomics Jul 2021Histidine phosphorylation is critically important in a variety of cellular processes including signal transduction, cell cycle, proliferation, differentiation, and...
Histidine phosphorylation is critically important in a variety of cellular processes including signal transduction, cell cycle, proliferation, differentiation, and apoptosis. It is estimated to account for 6% of all phosphorylated amino acids. However, due to the acid lability of the PN bond, the study of pHis lags far behind that of pSer, pThr, and pTyr. Recently, the development and use of pHis-specific antibodies and methodologies have led to a resurgence in the study of histidine phosphorylation. Although a considerable number of pHis proteins and sites have been discovered, most of them have not been manually curated and integrated to any databases. There is a lack of a data repository for pHis, and such work is expected to help further systemic studies of pHis. Thus, we present a comprehensive resource database of histidine phosphorylation (HisPhosSite) by curating experimentally validated pHis proteins and sites and compiling putative pHis sites with ortholog search. HisPhosSite contains 776 verified pHis sites and 2702 verified pHis proteins in 38 eukaryotic and prokaryotic species and 15,378 putative pHis sites and 10,816 putative pHis proteins in 1366 species. HisPhosSite provides rich annotations of pHis sites and proteins and multiple search engines (including motif search and BLAST search) for users to locate pHis sites of interest. HisPhosSite is available at http://reprod.njmu.edu.cn/hisphossite. SIGNIFICANCE: Histidine phosphorylation is involved in a variety of cellular processes as well as cancers, and it has been proved to be more common than previously thought. The HisPhosSite database was developed to collect pHis data from published literatures with experimental evidences. Unification of the identified pHis proteins and sites will give researchers an informative resource for histidine phosphorylation. HisPhosSite has a user-friendly interface with multiple search engines for users to locate pHis sites of interest. In addition, the database provides rich structural and functional annotations. HisPhosSite will help future studies and elucidation of the functions of histidine phosphorylation.
Topics: Antibodies; Histidine; Phosphorylation; Proteins; Signal Transduction
PubMed: 33984507
DOI: 10.1016/j.jprot.2021.104262 -
Phosphorylation status of pyruvate dehydrogenase in the mousebird Colius striatus undergoing torpor.Journal of Experimental Zoology. Part... Apr 2022Torpor is a heterothermic response that occurs in some animals to reduce metabolic expenditure. The speckled mousebird (Colius striatus) belongs to one of the few avian...
Torpor is a heterothermic response that occurs in some animals to reduce metabolic expenditure. The speckled mousebird (Colius striatus) belongs to one of the few avian taxa possessing the capacity for pronounced torpor, entering a hypometabolic state with concomitant decreases in body temperature in response to reduced food access or elevated thermoregulatory energy requirements. The pyruvate dehydrogenase complex (PDC) is a crucial site regulating metabolism by bridging glycolysis and the Krebs cycle. Three highly conserved phosphorylation sites are found within the E1 enzyme of the complex that inhibit PDC activity and reduce the flow of carbohydrate substrates into the mitochondria. The current study demonstrates a marked increase in S232 phosphorylation during torpor in liver, heart, and skeletal muscle of C. striatus. The increase in S232 phosphorylation during torpor was particularly notable in skeletal muscle where levels were ~49-fold higher in torpid birds compared to controls. This was in contrast to the other two phosphorylation sites (S293 and S300) which remained consistently phosphorylated regardless of tissue. The relevant PDH kinase (PDHK1) known to phosphorylate S232 was found to be substantially upregulated (~5-fold change) in the muscle during torpor as well as increasing moderately in the liver (~2.2-fold increase). Additionally, in the heart, a slight (~23%) decrease in total PDH levels was noted. Taken together the phosphorylation changes in PDH suggest that inhibition of the complex is a common feature across several tissues in the mousebird during torpor and that this regulation is mediated at a specific residue.
Topics: Animals; Birds; Oxidoreductases; Phosphorylation; Pyruvic Acid; Torpor
PubMed: 34951526
DOI: 10.1002/jez.2570