No abstract

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

Canonical Wnt signaling regulates many aspects of cellular physiology and tissue homeostasis during development and in adult organisms. In molecular terms, stimulation by Wnt ligands leads to the stabilization of β-catenin, its translocation to the nucleus, and stimulation of TCF (T-cell factor)-dependent transcription of target genes. This process is controlled at various stages by a number of regulatory proteins, including transcriptional activators and repressors. Here we demonstrate that the endosomal proteins APPL1 and APPL2 are novel activators of β-catenin/TCF-mediated transcription. APPL proteins are multifunctional adaptors and effectors of the small GTPase Rab5, which localize to a subpopulation of early endosomes but are also capable of nucleocytoplasmic shuttling. Overexpression of APPL1 or APPL2 protein stimulates the activity of β-catenin/TCF-dependent reporter construct, whereas silencing of APPL1 reduces it. Both APPL proteins interact directly with Reptin, a transcriptional repressor binding to β-catenin and HDAC1 (histone deacetylase 1), and this interaction was mapped to the pleckstrin homology domain of APPL1. Moreover, APPL proteins are present in an endogenous complex containing Reptin, β-catenin, HDAC1, and HDAC2. Overexpression of either APPL protein relieves Reptin-dependent transcriptional repression and correlates with the reduced amounts of HDACs and β-catenin associated with Reptin as well as with the lower levels of Reptin and HDAC1 on the promoters of β-catenin target genes. We propose that APPL proteins exert their stimulatory effects on β-catenin/TCF-dependent transcription by decreasing the activity of a Reptin-containing repressive complex.

No abstract

Several protein-tyrosine phosphatases (PTPs) have been implicated in the control of growth hormone receptor (GHR) signaling, but none have been shown to affect growth in vivo. We have applied a battery of molecular and cellular approaches to test a family-wide panel of PTPs for interference with GHR signaling. Among the subset of PTPs that showed activity in multiple readouts, we selected PTP-H1/PTPN3 for further in vivo studies and found that mice lacking the PTP-H1 catalytic domain show significantly enhanced growth over their wild type littermates. In addition, PTP-H1 mutant animals had enhanced plasma and liver mRNA expression of insulin-like growth factor 1, as well as increased bone density and mineral content. These observations point to a controlling role for PTP-H1 in modulating GHR signaling and systemic growth through insulin-like growth factor 1 secretion.

A B S T R A C TAccumulating evidence argues that many proteins governing membrane sorting during endocytosis participate also in nuclear signaling and transcriptional regulation, mostly by

No abstract

Multifunctional adaptor protein APPL1 [adaptor protein containing PH (pleckstrin homology) domain, PTB (phosphotyrosine binding) domain and leucine zipper motif] belongs to a growing group of endocytic proteins which actively participate in various stages of signalling pathways. Owing to its interaction with the small GTPase Rab5, APPL1 localizes predominantly to a subpopulation of early endosomes but is also capable of nucleocytoplasmic shuttling. Among its various binding partners, APPL1 was reported to associate with the nuclear co-repressor complex NuRD (nucleosome remodelling and deacetylase), containing both nucleosome remodelling and HDAC (histone deacetylase) activities, but the biochemical basis or functional relevance of this interaction remained unknown. Here we characterized the binding between APPL1 and NuRD in more detail, identifying HDAC2 as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD complex containing enzymatically active HDAC2 but not HDAC1 as the only deacetylase. However, the cellular levels of HDAC1 can regulate the extent of APPL1–NuRD interactions, which in turn modulates the nucleocytoplasmic distribution of APPL1. Increased binding of APPL1 to NuRD upon silencing of HDAC1 promotes the nuclear localization of APPL1, whereas HDAC1 overexpression exerts an opposite effect. Moreover, we also uncovered a NuRD-independent interaction of APPL1 with HDAC1. APPL1 overexpression affects the composition of the HDAC1-containing NuRD complex and the expression of HDAC1 target p21WAF1/CIP1. Cumulatively, these data reveal a surprising complexity of APPL1 interactions with HDACs, with functional consequences for the modulation of gene expression. In a broader sense, these results contribute to an emerging theme of endocytic proteins playing alternative roles in the cell nucleus.