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To assess potential tyrosine phosphorylation events in H2A, we individually mutated every tyrosine residue in H2A to phenylalanine and expressed the mutants in 293T cells. Mutation of Tyr 39 and [...], Post-translational histone modifications have a critical role in regulating transcription, the cell cycle, DNA replication and DNA damage repair (1). The identification of new histone modifications critical for transcriptional regulation at initiation, elongation or termination is of particular interest. Here we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals. This regulation is based on the phosphorylation of a highly conserved tyrosine residue, Tyr 57, in histone H2A and is mediated by the unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of Tyr 57 in H2A in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA-polymerase-II-transcribed coding genes and active enhancers. Mutation of Tyr 57 causes a loss of H2B mono-ubiquitination as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and the H2A(Y57F) mutation enhance H2B deubiquitination activity of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA complex during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A.

Autophagy is the major cellular pathway by which macromolecules are degraded, and amino acid depletion powerfully activates autophagy. MAP4K3, or germinal-center kinase-like kinase, is required for robust cell growth in response to amino acids, but the basis for MAP4K3 regulation of cellular metabolic disposition remains unknown. Here we identify MAP4K3 as an amino acid-dependent regulator of autophagy through its phosphorylation of transcription factor EB (TFEB), a transcriptional activator of autophagy, and through amino acid starvation-dependent lysosomal localization of MAP4K3. We document that MAP4K3 physically interacts with TFEB and MAP4K3 inhibition is sufficient for TFEB nuclear localization, target gene transactivation, and autophagy, even when mTORC1 is activated. Moreover, MAP4K3 serine 3 phosphorylation of TFEB is required for TFEB interaction with mTORC1-Rag GTPase-Ragulator complex and TFEB cytosolic sequestration. Our results uncover a role for MAP4K3 in the control of autophagy and reveal MAP4K3 as a central node in nutrient-sensing regulation. [ABSTRACT FROM AUTHOR]

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated fromConus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, twoγ-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed ofN-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen,β-d-Gal-(1→3)-α-d-GalNAc, one of the most common O-linked glycan structures, identified previously in anotherConusglycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = β-d-Gal-(1→3)-α-d-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional1H homonuclear and13C-1H heteronuclear NMR spectroscopy of native tx5a isolated fromConus textilewas then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but ratherα-d-Gal-(1→3)-α-d-GalNAc. [ABSTRACT FROM AUTHOR]

A study was performed to characterize hand extremity doses received by a laboratory researcher performing a metabolic cell labeling procedure using 10-cm-diameter cell culture dishes containing GBq amounts of 32P-orthophosphate. Specifically, the optimal location for placement of thermoluminescent dosimeters on the extremities was determined as was the phase (time frame) during the cell labeling procedure when the highest dose was received. The cell labeling procedure was divided into four phases. Thermoluminescent dosimeters were placed on three fingers of each hand at the start of each phase and after the completion of that phase, collected, and sent to the processor.It was determined that in the case of this right-handed worker, the left-hand index finger should be used for determining the maximum extremity dose. The time frame during which the highest extremity dose was received was the cell lysis phase. Acrylic shielding was fabricated, which significantly reduced worker extremity dose. [ABSTRACT FROM AUTHOR]

Identities of functional pSer/Thr.Pro protein substrates of the PIN1 prolyl isomerase and its effects on downstream signaling in bladder carcinogenesis remain largely unknown. Phenotypically, we found that PIN1 positively regulated bladder cancer cell proliferation, cell motility and urothelium clearance capacity in vitro and controlled tumor growth and potential metastasis in vivo. Mechanistically, we observed a negative enrichment of SREBP2-driven cholesterol metabolism pathways and a decrease in free/total cholesterol levels in PIN1-knockout bladder cancer cells. Moreover, we showed that PIN1 interacted with SREBP2 following its phosphorylation by the JNK MAP kinase at Ser455, which lies near the Site-2 cleavage site that generates the active, nuclear-form of SREBP2. Therapeutically, a combination of the sulfopin PIN1 covalent inhibitor and the simvastatin HMGCoA reductase inhibitor suppressed cell proliferation in vitro and tumor growth in vivo synergistically. Together, these findings emphasize that PIN1 can act as a driver and potential therapeutic target in bladder cancer.

Histidine phosphorylation (pHis) is a non-canonical post-translational modification (PTM) that is historically understudied due to a lack of robust reagents that are required for its investigation, such as high affinity pHis-specific antibodies. Engineering pHis-specific antibodies is very challenging due to the labile nature of the phosphoramidate (P-N) bond and the stringent requirements for selective recognition of the two isoforms, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Here, we present a strategy for in vitro engineering of antibodies for detection of native 3-pHis targets. Specifically, we humanized the rabbit SC44-8 anti-3-pTza (a stable 3-pHis mimetic) mAb into a scaffold (herein referred to as hSC44) that was suitable for phage display. We then constructed six unique Fab phage-displayed libraries using the hSC44 scaffold and selected high affinity 3-pHis binders. Our selection strategy was carefully designed to enrich antibodies that bound 3-pHis with high affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32F L , the best engineered antibody, has an ~10-fold higher affinity for 3-pHis than the parental hSC44. Eleven new Fab structures, including the first reported antibody-pHis peptide structures were solved by X-ray crystallography. Structural and quantum mechanical calculations provided molecular insights into 3-pHis and 3-pTza discrimination by different hSC44 variants and their affinity increase obtained through in vitro engineering. Furthermore, we demonstrate the utility of these newly developed high-affinity 3-pHis-specific antibodies for recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Overall, our work describes a general method for engineering PTM-specific antibodies and provides a set of novel antibodies for further investigations of the role of 3-pHis in cell biology., Competing Interests: Competing Interest Statement: Authors declare no competing interests.

The pursuit of scientific knowledge has been considerably advanced by the use of biochemical molecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules, and/or the radiolabeled products that result from biochemical reactions in which the parent molecule was involved, can be traced using a variety of instruments that detect radioactivity. This appendix begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a molecular biology laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detection and imaging methods used in experimental analysis are reviewed. Finally, an outline of an orderly response to a spill of radioactive material is presented.

The pursuit of scientific knowledge has been considerably advanced by the use of biochemical molecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules, and/or the radiolabeled products that result from biochemical reactions in which the parent molecule was involved, can be traced using a variety of instruments that detect radioactivity. This appendix begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a molecular biology laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detection and imaging methods used in experimental analysis are reviewed. Finally, an outline of an orderly response to a spill of radioactive material is presented., (© 2008 by John Wiley & Sons, Inc.)

The pursuit of scientific knowledge has been considerably advanced by the use of a wide variety of hazardous substances, including radioisotopes, hazardous chemicals, and biohazards. This appendix addresses each of these classes of hazards and primary safety precautions that need to be considered., (Copyright 2008 by John Wiley & Sons, Inc.)

The pursuit of scientific knowledge has been considerably advanced by the use of biochemical molecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules, and/or the radiolabeled products that result from biochemical reactions in which the parent molecule was involved, can be traced using a variety of instruments that detect radioactivity. This appendix begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a molecular biology laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detection and imaging methods used in experimental analysis are reviewed. Finally, an outline of an orderly response to a spill of radioactive material is presented., ((c) 2007 by John Wiley & Sons, Inc.)

The use of radioisotopes to label specific molecules in a defined way has greatly furthered the discovery and dissection of biochemical pathways. The development of methods to synthesize such tagged biological compounds inexpensively on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries. In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses handling, storage, and disposal of the isotopes most frequently used in biological research.