Conventional molecular dynamics (MD) simulations using the GPU-enabled CUDA version of the pmemd executable (pmemd.cuda) in AMBER were applied to explore the structure and dymanics of MAPK, ERK2. The role of dual phosphorylation was explored by comparing 0P-ERK2 with 2P-ERK2 (two phosphate groups added at T183 and Y185). Both 0P-ERK2 and 2P-ERK2 models were treated with the ff19SB forcefield immersed in a solution of 0.15 M NaCl in OPC water. The results showed that the A-loop can adopt multiple long-lived (>5 microseconds) conformational states. A set of primary and secondary seeds were used to explore these novel states of the activation loop. Analysis scripts, dataframes, and all trajectories (stripped of explicit water and NaCl ions) are provided here to enhance the reproducibility of this complex study and aid future studies. Trajectories As described in the associated publication, individual trajectories were run for (5-25) microseconds and totaled 727 microseconds. Multiple primary seeds (285 K, 300 K, 315 K, and 330 K) and secondary trajectory seeds (300 K) were run; all are available in this data publication. All frames for each trajectory were aligned to backbone atoms of residues 10-161 and 182-343 of the minimized 2ERK X-ray structures (0P and 2P). Modeled residue numbers are 1-353 and correspond to residues 6-358 of 2ERK and 5UMO (rat sequence numbering). The model built from 2Y9Q, which is a human kinase, was converted to correspond to the rat-derived models (2ERK, 5UMO) as described in the associated publication. Most primary seeds are greater than 10 microseconds while most secondary seeds are 5.7 microseconds. Angstrom units (0.1 nm) are used for trajectory coordinate storage and analysis. Three sets of trajectories are provided for convenience: 1. The canonical set of trajectories are netcdf files (extension .nc) that are stripped of NaCl and water molecules and down-sampled to 2.5 ns between frames. 2. The set of trajectory seeds with hydrogens removed and further down-sampling to 250 ns between frames is provided as DCD trajectory files. This is the smallest download that will be most convenient for visualization and initial development. 3. Frames from the 300 K trajectories collated by the A-loop states. These trajectories should be treated as collections of frames without regard for any time information stored. Structures from RCSB.org The associated publication analyzed the crystal packing environment of the activation loop. The structural data were collected in November 2021. All pdb files downloaded at that time are included in this data publication to aid the reproducibility of the analysis. The RCSB (Research Collaboratory for Structural Bioinformatics, https://www.rcsb.org) was the source of information and should be used directly for current versions of the associated entries. The RCSB entries pdbid 2ERK, pdbid 5UMO, and pdbid 2Y9Q were the structures used to build initial models for MD simulations. Scripts Representative scripts are provided to aid future work. See README.md Units Q-Aloop values are fractions. RMSD, distances, and coordinates are all reported in Angstroms (0.1 nm). Angles are reported in degrees. NOTE: Trade names are provided only to specify the source of information and procedures adequately and do not imply endorsement by the National Institute of Standards and Technology. Similar products by other developers may be found to work as well or better.

Background The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the β-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1. Results Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM). Conclusions The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal β-propeller blades of RACK1 in a manner akin to the interaction of the helical G-γ signal transducing protein with the β-propeller protein, G-β.

Background In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus. Results Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene). Conclusions We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.

ToxCast bioactivity data and model predictions for the estrogen receptor (ER) and androgen receptor (AR) pathways were obtained from the inks provided. This dataset is associated with the following publication: Lizarraga, L., J. Dean, J. Kaiser, S. Wesselkamper, J. Lambert, and J. Zhao. A Case Study on the Application of An Expert-driven Read-Across Approach in Support of Quantitative Risk Assessment of p,p’-Dichlorodiphenyldichloroethane. REGULATORY TOXICOLOGY AND PHARMACOLOGY. Elsevier Science Ltd, New York, NY, USA, 103: 301-313, (2019).

Background In most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR. Results To further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP. Conclusion These studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

Multiplex ELisa data, MTF-1 and western blot. This dataset is associated with the following publication: Bruno, M., J. Ross, and Y. Ge. Proteomic Responses of BEAS-2B Cells to Nontoxic and Toxic Chromium: Protein Indicators of Cytotoxicity Conversion. TOXICOLOGY LETTERS. Elsevier Science Ltd, New York, NY, USA, 264: 59-70, (2016).