@article{1299940,

title = {Investigation of N-Terminal Phospho-Regulation of~Uracil DNA Glycosylase Using Protein Semisynthesis},

author = {Brian P Weiser and James T Stivers and Philip A Cole},

doi = {10.1016/j.bpj.2017.06.016},

issn = {1542-0086},

year  = {2017},

date = {2017-07-01},

journal = {Biophys J},

volume = {113},

number = {2},

pages = {393-401},

abstract = {Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that prevents the stable incorporation of deoxyuridine monophosphate into DNA in the form of U/A basepairs. During S-phase, UNG2 remains associated with the replication fork through its interactions with two proteins, Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which are critical for DNA replication and repair. In this work, we used protein semisynthesis and fluorescence anisotropy assays to explore the interactions of UNG2 with PCNA and RPA and to determine the effects of two UNG2 phosphorylation sites (Thr6 and Tyr8) located within its PCNA-interacting motif (PIP-box). In binding assays, we found that phosphorylation of Thr6 or Tyr8 on UNG2 can impede PCNA binding without affecting UNG2 catalytic activity or its RPA interaction. Our data also suggests that unmodified UNG2, PCNA, and RPA can form a ternary protein complex. We propose that the UNG2 N-terminus may serve as a flexible scaffold to tether PCNA and RPA at the replication fork, and that post-translational modifications on the UNG2 N-terminus disrupt formation of the PCNA-UNG2-RPA protein complex.},

keywords = {Catalysis, DNA Glycosylases, Electrospray Ionization, Escherichia coli, Humans, Mass, Mutation, Phosphorylation, Proliferating Cell Nuclear Antigen, Protein Binding, Protein Domains, Protein Stability, Replication Protein A, Spectrometry},

pubstate = {published},

tppubtype = {article}

}

@article{1299944,

title = {A Tunable Brake for HECT Ubiquitin Ligases},

author = {Zan Chen and Hanjie Jiang and Wei Xu and Xiaoguang Li and Daniel R Dempsey and Xiangbin Zhang and Peter Devreotes and Cynthia Wolberger and L Mario Amzel and Sandra B Gabelli and Philip A Cole},

doi = {10.1016/j.molcel.2017.03.020},

issn = {1097-4164},

year  = {2017},

date = {2017-05-01},

journal = {Mol Cell},

volume = {66},

number = {3},

pages = {345-357.e6},

abstract = {The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.},

keywords = {Allosteric Regulation, Endosomal Sorting Complexes Required for Transport, Enzyme Activation, Enzyme Stability, HeLa Cells, Humans, Models, Molecular, Mutation, Nedd4 Ubiquitin Protein Ligases, Phosphorylation, Post-Translational, Protein Domains, Protein Processing, Proteolysis, Repressor Proteins, Structure-Activity Relationship, Transfection, Ubiquitin-Protein Ligases},

pubstate = {published},

tppubtype = {article}

}

@article{1299976,

title = {How IGF-1 activates its receptor},

author = {Jennifer M Kavran and Jacqueline M McCabe and Patrick O Byrne and Mary Katherine Connacher and Zhihong Wang and Alexander Ramek and Sarvenaz Sarabipour and Yibing Shan and David E Shaw and Kalina Hristova and Philip A Cole and Daniel J Leahy},

doi = {10.7554/eLife.03772},

issn = {2050-084X},

year  = {2014},

date = {2014-09-01},

journal = {Elife},

volume = {3},

abstract = {The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.},

keywords = {Amino Acid Sequence, Animals, Conserved Sequence, HEK293 Cells, Humans, IGF Type 1, Insulin, Insulin-Like Growth Factor I, Ligands, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Phosphorylation, Protein Binding, Protein Multimerization, Protein Structure, Receptor, Tertiary},

pubstate = {published},

tppubtype = {article}

}

@article{1299974,

title = {Catalytic mechanisms and regulation of protein kinases},

author = {Z. Wang and P. A. Cole},

doi = {10.1016/B978-0-12-397918-6.00001-X},

issn = {1557-7988},

year  = {2014},

date = {2014-00-00},

journal = {Methods Enzymol.},

volume = {548},

pages = {1-21},

abstract = {Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology.},

keywords = {Adenosine Triphosphate, Animals, Biocatalysis, Humans, Models, Molecular, Mutation, Phosphorylation, Post-Translational, Protein Conformation, Protein Kinase Inhibitors, Protein Kinases, Protein Processing, Substrate Specificity},

pubstate = {published},

tppubtype = {article}

}