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Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas (1) and cytogenetically normal acute myeloid leukaemias (AML) (2). These alterations are gain-of-function mutations in that they drive the synthesis of the 'oncometabolite' R-2-hydroxyglutarate (2HG) (3). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML., IDH1/IDH2 mutations typically produce mutant enzymes with aberrant activity. Whereas wild-type (WT) Idh proteins metabolize isocitrate and [NADP.sup.+] to yield α-ketoglutarate (αKG) and NADPH, mutant Idh proteins convert αKG into [...]

The [alpha]-, [beta], and [gamma]-protocadherins (Pcdh[alpha], Pcdh[beta], and Pcdh[gamma]) comprise a large family of single-pass transmembrane proteins predominantly expressed in the nervous system. These proteins contain six cadherin-like extracellular domains, and proteolysis of Pcdh[alpha] and Pcdh[gamma] by the [gamma]-secretase complex releases their intracellular domains into the cytoplasm where they may function locally and/or enter the nucleus and affect gene expression. Thus, cleavage of Pcdhs may function to link intercellular contacts and intracellular signaling. Here we report that shedding of the Pcdh[alpha] extracellular domain and subsequent processing by [gamma]-secretase require endocytosis and that Pcdhs interact with the regulator of vesicular sorting ESCRT-0 in undifferentiated cells. We also find that the accumulation of Pcdh cleavage products is regulated during development. Differentiation leads to an increase in the interactions between Pcdh proteins and a decrease in the accumulation of cleavage products. We conclude that Pcdh processing requires endocytosis and that the level of cleavage products is regulated during neuronal differentiation. [gamma]-secretase | hepatocyte growth factor-regulated tyrosine kinase substrate | neurodevelopment | protein-protein interactions | proteolysis doi/ 10.1073/pnas.1013105107

The clustered protocadherins (Pcdhs) are a large family of cadherin-like transmembrane proteins expressed in the nervous system. Stochastic expression of Pcdh genes and alternative splicing of their pre-mRNAs have the potential to generate enormous protein diversity at the cell surface of neurons. At present, the regulation and function of Pcdh proteins are largely unknown. Here, we show that Pcdhs form a heteromeric signaling complex(es), consisting of multiple Pcdh isoforms, receptor tyrosine kinases, phosphatases, and cell adhesion molecules. In particular, we find that the receptor tyrosine kinase rearranged during transformation (Ret) binds to Pcdhs in differentiated neuroblastoma cells and is required for stabilization and differentiation-induced phosphorylation of Pcdh proteins. In addition, the Ret ligand glial cell line-derived neurotrophic factor induces phosphorylation of Pcdhy in motor neurons and phosphorylation of Pcdh[alpha] and Pcdh[gamma] in sympathetic neurons. Conversely, Pcdh proteins are also required for the stabilization of activated Ret in neuroblastoma cells and sympathetic ganglia. Thus, Pcdhs and Ret are functional components of a phosphorylation-dependent signaling complex. intracellular domain | signal transduction | TAP tag | protein-protein interactions | protein purification doi/ 10.1073/pnas.1007182107