Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5′ end of Xist.

Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory "molecular brake" mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSDlocalized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission. Neurons are highly polarized cells, specializing in the reception of numerous, independent signal inputs and rapid integration of these inputs into an electrochemical response. The major sites of signal input are synapses, which are highly ordered cell junctions formed between two neurons and are typically unidirectional in fast excitatory chemical neurotransmission in the mammalian CNS. The response to neurotransmitter (NT) 1 release at the synapse is provided by a protein matrix of NT receptors and supporting proteins collectively known as the postsynaptic density (PSD) (for review, see Refs. 1-3). The PSD has several proposed functions including: signal amplification, cytoskeletal anchorage, biochemical signaling regulation, and NT receptor clustering (1, 4 -6).Changes in size and composition of the PSD correlate with changes in synaptic strength (7,8), including alterations that are stably maintained such as long-term potentiation (LTP), a physiologically relevant increase in synaptic efficacy and a model for learning and memory (9, 10). Therefore, an understanding of the protein composition of the PSD is a prerequisite for modeling the molecular interactions regulating synaptic strength.The structure of PSDs purified from rodent brains using gradient centrifugation and Triton X-100 extraction has been shown by electron microscopy (EM) to be virtually identical to the "in vivo" PSD structure (4, 11). Gel electrophoresis, enzymatic activity assays, and EM experiments have demonstrated that this procedure yields a highly pure, membranefree PSD fraction (11,12). Recent proteomic studies have investigated the composition of the PSD by SDS-PAGE or two-dimensional gel electrophoresis (2DE) coupled with MS (13-16). Li et al. (16) also performed shotgun proteomics using cysteine-containing peptides selected using ICAT techniques. However, each of these investigations identified less than one-third of previously described and biochemically confirmed PSD components, pointing to limitations in the techniques used. A recent paper by Yoshimura et al. (17) reports the identification by mass spectrometry of 492 proteins in the PSD, which suggests that the PSD is more ...

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity contributing to myeloproliferative neoplasms (MPNs). Here, we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2, Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Importantly, different MPN mutations were found to abrogate JH2 activity in cells, and in MPN (V617F) patient cells, phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2.