It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell receptor (TCR) triggering, but how segregation would occur and whether it can initiate signaling is unclear. Using structural and biophysical analysis we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of new structures characterized by spontaneous sub-micron scale CD45 and kinase segregation, called ‘close-contacts’, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the unexpectedly potent signaling effects of local CD45 and kinase segregation. TCR ligands could heighten signaling simply by holding receptors in close-contacts.

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPs). Here we report that RPTPs acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPs ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPs and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.

Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IB␣ were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARDcontaining proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.NF-B ͉ 2-oxoglutarate-dependent dioxygenase ͉ protein hydroxylation C ells react to variation in oxygen availability with adaptive responses that involve changes in most basic cellular functions. Analysis of the transcriptional component of this response has defined pathways that regulate hypoxia-inducible factor (HIF) by posttranslational hydroxylation of specific residues. HIF is an ␣͞␤ heterodimer that binds hypoxia response elements in a range of hypoxia-inducible genes (for review, see ref. 1). Regulation is mediated by the ␣-subunits and involves dual mechanisms controlling both the abundance and activity of the protein. Thus, hydroxylation of specific prolyl residues promotes interaction with the von Hippel-Lindau E3 ligase and hence proteolysis, whereas hydroxylation of a C-terminal Asn residue blocks recruitment of the coactivators p300͞CBP. The prolyl and asparaginyl hydroxylase enzymes that catalyze these reactions are 2-oxoglutarate (2-OG) and Fe(II)-dependent dioxygenases that couple the oxidative decarboxylation of 2-OG with oxidation of peptidyl substrates. Dioxygen is an obligate cosubstrate, and reductions in the rate of hydroxylation during hypoxia allow HIF-␣ to escape VHLmediated destruction and to activate transcription (for reviews, see refs. 2 and 3).HIF prolyl hydroxylation is catalyzed by three enzymes, PHD1, -2, and -3 (equivalent to EGLN2, -1, and -3 and HPH-3, -2, and -1). HIF Asn hydroxylation is catalyzed by a more distantly related 2-OG-dependent dioxygenase, factor inhibiting HIF (FIH) (for reviews, see refs. 2 and 3). A key question raised by these findings is whether the roles of all four dioxygenases are specific to HIF regulation, or whether one or more have alternative substrates. Several studies have identified proteins that interact to modulate HIF hydroxylase activity (4) or ...