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Nature Chemical Biology Jul 2015Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of...
Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access--including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
Topics: Amino Acyl-tRNA Synthetases; Base Sequence; Codon, Terminator; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genetic Code; Models, Molecular; Molecular Sequence Data; NIMA-Related Kinases; Nucleic Acid Conformation; Phosphorylation; Phosphoserine; Protein Engineering; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Recombinant Proteins; Ubiquitin
PubMed: 26030730
DOI: 10.1038/nchembio.1823 -
International Journal of Molecular... May 2018Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of β-catenin, and results in the enhanced accumulation of the Cx43...
Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of β-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, β-catenin co-localizes with the Cx43 protein itself as part of a complex at the gap junction plaque. Work from several labs have also shown indirect evidence of this interaction via reciprocal co-immunoprecipitation. Our goal for the current study was to identify whether β-catenin directly interacts with Cx43, and if so, the location of that direct interaction. Identifying residues involved in direct protein⁻protein interaction is of importance when they are correlated to the phosphorylation of Cx43, as phosphorylation can modify the binding affinities of Cx43 regulatory protein partners. Therefore, combining the location of a protein partner interaction on Cx43 along with the phosphorylation pattern under different homeostatic and pathological conditions will be crucial information for any potential therapeutic intervention. Here, we identified that β-catenin directly interacts with the Cx43 carboxyl-terminal domain, and that this interaction would be inhibited by the Src phosphorylation of Cx43CT residues Y265 and Y313.
Topics: Amino Acid Sequence; Animals; Circular Dichroism; Connexin 43; Humans; Magnetic Resonance Spectroscopy; Phosphorylation; Phosphoserine; Protein Binding; Protein Domains; Protein Structure, Secondary; Rats; Surface Plasmon Resonance; beta Catenin
PubMed: 29882937
DOI: 10.3390/ijms19061562 -
Journal of Lipid Research Mar 2003In eukaryotes, phosphatidylserine (PtdSer) can serve as a precursor of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho), which are the major cellular... (Review)
Review
In eukaryotes, phosphatidylserine (PtdSer) can serve as a precursor of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho), which are the major cellular phospholipids. PtdSer synthesis originates in the endoplasmic reticulum (ER) and its subdomain named the mitochondria-associated membrane (MAM). PtdSer is transported to the mitochondria in mammalian cells and yeast, and decarboxylated by PtdSer decarboxylase 1 (Psd1p) to form PtdEtn. A second decarboxylase, Psd2p, is also found in yeast in the Golgi-vacuole. PtdEtn produced by Psd1p and Psd2p can be transported to the ER, where it is methylated to form PtdCho. Organelle-specific metabolism of the aminoglycerophospholipids is a powerful tool for experimentally following lipid traffic that is now enabling identification of new proteins involved in the regulation of this process. Genetic and biochemical experiments demonstrate that transport of PtdSer between the MAM and mitochondria is regulated by protein ubiquitination, which affects events at both membranes. Similar analyses of PtdSer transport to the locus of Psd2p now indicate that a membrane-bound phosphatidylinositol transfer protein and the C2 domain of Psd2p are both required on the acceptor membrane for efficient transport of PtdSer. Collectively, these recent findings indicate that novel multiprotein assemblies on both donor and acceptor membranes participate in interorganelle phospholipid transport.
Topics: Animals; Biological Transport; Cell Membrane; Glycerophospholipids; Mitochondria; Phosphoserine; Ubiquitin
PubMed: 12562848
DOI: 10.1194/jlr.R200020-JLR200 -
Journal of Leukocyte Biology Feb 2017The common FcRγ, an immunoreceptor tyrosine-based activation motif (ITAM)- containing adaptor protein, associates with multiple leukocyte receptor complexes and...
The common FcRγ, an immunoreceptor tyrosine-based activation motif (ITAM)- containing adaptor protein, associates with multiple leukocyte receptor complexes and mediates signal transduction through the ITAM in the cytoplasmic domain. The presence of multiple serine and threonine residues within this motif suggests the potential for serine/threonine phosphorylation in modulating signaling events. Single-site mutational analysis of these residues in RBL-2H3 cells indicates that each may contribute to net FcRγ-mediated signaling, and mass spectrometry of WT human FcRγ from receptor-stimulated cells shows consistent preferential phosphorylation of the serine residue at position 51. Immunoblot analysis, mass spectrometry, and mutational analyses showed that phosphorylation of serine 51 in the 7-residue spacer between the 2 YxxL sequences regulates FcRγ signaling by inhibiting tyrosine phosphorylation at the membrane proximal Y47 position of the ITAM, but not phosphorylation at position Y58. This inhibition results in reduced Syk recruitment and activation. With in vitro kinase assays, PKC-δ and PKA show preferential phosphorylation of S51. Serine/threonine phosphorylation of the FcRγ ITAM, which functions as an integrator of multiple signaling elements, may explain in part the contribution of variants in PKC-δ and other PKC isoforms to some autoimmune phenotypes.
Topics: Amino Acid Motifs; Cyclic AMP-Dependent Protein Kinases; Humans; Phosphorylation; Phosphoserine; Protein Kinase C; Receptors, Fc; Signal Transduction; Syk Kinase; Threonine
PubMed: 27630214
DOI: 10.1189/jlb.2AB0516-228R -
Traffic (Copenhagen, Denmark) Aug 2017HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets...
HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP-1 and AP-2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α-chain. CD8-VpuCD was rapidly endocytosed in a clathrin- and AP-2-dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β-TrCP binding site and a leucine-based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV-dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP-2 and showed that this is enhanced by serine-phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP-2 and AP-1; this interaction was dependent on serine-phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.
Topics: Adaptor Proteins, Vesicular Transport; Endocytosis; HeLa Cells; Human Immunodeficiency Virus Proteins; Humans; Phosphorylation; Phosphoserine; Viral Regulatory and Accessory Proteins
PubMed: 28504462
DOI: 10.1111/tra.12495 -
The Journal of Cell Biology Feb 2022In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long...
In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription "factories," and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody's specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.
Topics: Cell Nucleus; Cell Survival; HeLa Cells; Humans; Interphase; Molecular Imaging; Phosphorylation; Phosphoserine; RNA Polymerase II; RNA Probes; Transcription, Genetic
PubMed: 34854870
DOI: 10.1083/jcb.202104134 -
Proceedings of the National Academy of... Apr 2013Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of...
Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn(2+)-dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase activity). The best deoxyribozyme decreases the half-life for phosphoserine hydrolysis from as high as >10(10) y to <1 h. The phosphatase activity also occurs with nonpeptidic substrates but with reduced efficiency, indicating a preference for phosphopeptides. The newly identified deoxyribozymes can function with multiple turnover using free peptide substrates, have activity in the presence of human cell lysate or BSA, and catalyze dephosphorylation of a larger protein substrate, suggesting broader application of DNA catalysts as artificial phosphatases.
Topics: Catalysis; Chromatography, High Pressure Liquid; DNA, Catalytic; Electrophoresis, Polyacrylamide Gel; Half-Life; Humans; Hydrolysis; In Vitro Techniques; Mass Spectrometry; Oligonucleotides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphoserine; Phosphotyrosine; Rosaniline Dyes
PubMed: 23509279
DOI: 10.1073/pnas.1221946110 -
Scientific Reports Apr 2021CHK1 is a crucial DNA damage checkpoint kinase and its activation, which requires ATR and RAD17, leads to inhibition of DNA replication and cell cycle progression....
CHK1 is a crucial DNA damage checkpoint kinase and its activation, which requires ATR and RAD17, leads to inhibition of DNA replication and cell cycle progression. Recently, we reported that SMG7 stabilizes and activates p53 to induce G arrest upon DNA damage; here we show that SMG7 plays a critical role in the activation of the ATR-CHK1 axis. Following genotoxic stress, SMG7-null cells exhibit deficient ATR signaling, indicated by the attenuated phosphorylation of CHK1 and RPA32, and importantly, unhindered DNA replication and fork progression. Through its 14-3-3 domain, SMG7 interacts directly with the Ser635-phosphorylated RAD17 and promotes chromatin retention of the 9-1-1 complex by the RAD17-RFC, an essential step to CHK1 activation. Furthermore, through maintenance of CHK1 activity, SMG7 controls G-M transition and facilitates orderly cell cycle progression during recovery from replication stress. Taken together, our data reveals SMG7 as an indispensable signaling component in the ATR-CHK1 pathway during genotoxic stress response.
Topics: Amino Acid Sequence; Ataxia Telangiectasia Mutated Proteins; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Checkpoint Kinase 1; Chromatin; DNA Damage; DNA Replication; HCT116 Cells; Humans; Phosphorylation; Phosphoserine; Protein Binding
PubMed: 33820915
DOI: 10.1038/s41598-021-86957-x -
Acta Neuropathologica Communications Mar 2016A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in...
A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in which neurons not directly affected by the disease are denervated. These transneuronal effects on the network contribute considerably to the clinical symptoms. Since denervated neurons are viable, they are attractive targets for intervention. Therefore, we studied the role of Sphingosine-1-phosphate (S1P)-receptor signaling, the target of Fingolimod (FTY720), in denervation-induced dendritic atrophy. The entorhinal denervation in vitro model was used to assess dendritic changes of denervated mouse dentate granule cells. Live-cell microscopy of GFP-expressing granule cells in organotypic entorhino-hippocampal slice cultures was employed to follow individual dendritic segments for up to 6 weeks after deafferentation. A set of slice cultures was treated with FTY720 or the S1P-receptor (S1PR) antagonist VPC23019. Lesion-induced changes in S1P (mass spectrometry) and S1PR-mRNA levels (laser microdissection and qPCR) were determined. Denervation caused profound changes in dendritic stability. Dendritic elongation and retraction events were markedly increased, resulting in a net reduction of total dendritic length (TDL) during the first 2 weeks after denervation, followed by a gradual recovery in TDL. These changes were accompanied by an increase in S1P and S1PR1- and S1PR3-mRNA levels, and were not observed in slice cultures treated with FTY720 or VPC23019. We conclude that inhibition of S1PR signaling prevents dendritic destabilization and denervation-induced dendrite loss. These results suggest a novel neuroprotective effect for pharmaceuticals targeting neural S1PR pathways.
Topics: Animals; Animals, Newborn; Atrophy; Calcium-Binding Proteins; Dendrites; Denervation; Entorhinal Cortex; Fingolimod Hydrochloride; Gene Expression Regulation; Immunosuppressive Agents; In Vitro Techniques; Mice; Mice, Transgenic; Neurons; Organ Culture Techniques; Perforant Pathway; Phosphoserine; Receptors, Lysosphingolipid; Time Factors; Up-Regulation
PubMed: 27036416
DOI: 10.1186/s40478-016-0303-x -
Cell Death & Disease May 2020DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand...
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks, and its activity is strictly controlled by DNA-PKcs phosphorylation. The ubiquitin-like protein, NEDD8 is involved in regulation of DNA damage response, but it remains mysterious whether and how NEDD8-related neddylation affects DNA-PKcs and the NHEJ process. Here, we show that DNA-PKcs is poly-neddylated at its kinase domain. The neddylation E2-conjugating enzyme UBE2M and E3 ligase HUWE1 (HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1) are responsible for the DNA-PKcs neddylation. Moreover, inhibition of HUWE1-dependent DNA-PKcs neddylation impairs DNA-PKcs autophosphorylation at Ser2056. Finally, depletion of HUWE1-dependent DNA-PKcs neddylation reduces the efficiency of NHEJ. These studies provide insights how neddylation modulates the activity of NHEJ core complex.
Topics: Cell Line; DNA Damage; DNA End-Joining Repair; DNA-Activated Protein Kinase; Humans; NEDD8 Protein; Phosphorylation; Phosphoserine; Protein Domains; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases
PubMed: 32457294
DOI: 10.1038/s41419-020-2611-0