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This article expands on crystal structure data for human H-RAS with mutations at position Y137, briefly described in a paper on the effects of phosphorylation of Y137 by ABL kinases (Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding, published in the FASEB Journal [1]). The crystal structures of the Y137E mutant (phosphorylation mimic) and of the Y137F mutant (without the hydroxyl group where phosphorylation occurs) were deposited in the Protein Data Bank with PDB codes 4XVQ (H-RASY137E) and 4XVR (H-RASY137F). This article includes details for expression and purification of RAS and its mutants with no affinity tags, in vitro exchange of guanine nucleotides, protein crystallization, X-ray data collection and structure refinement. Keywords: RAS GTPase, Protein purification, Nucleotide exchange, X-ray crystal structures

© 2015 Ting et al. Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET)-based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.

Homeostatic plasticity is thought to play an important role in maintaining the stability of neuronal circuits. During one form of homeostatic plasticity, referred to as synaptic scaling, activity blockade leads to a compensatory increase in synaptic transmission by stimulating in dendrites the local translation and synaptic insertion of the AMPA receptor subunit GluR1. We have previously shown that all-trans retinoic acid (RA) mediates activity blockade-induced synaptic scaling by activating dendritic GluR1 synthesis and that this process requires RARα, a member of the nuclear RA receptor family. This result raised the question of where RARα is localized in dendrites and whether its localization is regulated by RA and/or activity blockade. Here, we show that activity blockade or RA treatment in neurons enhances the concentration of RARα in the dendritic RNA granules and activates local GluR1 synthesis in these RNA granules. Importantly, the same RNA granules that contain RARα also exhibit an accumulation of GluR1 protein but with a much slower time course than that of RARα, suggesting that the former regulates the latter. Taken together, our results provide a direct link between dendritically localized RARα and local GluR1 synthesis in RNA granules during RA-mediated synaptic signaling in homeostatic synaptic plasticity.