c-Cbl and Cbl-b Ligases Mediate 17-Allylaminodemethoxygeldanamycin-induced Degradation of Autophosphorylated Flt3 Kinase with Internal Tandem Duplication through the Ubiquitin Proteasome Pathway

The class III receptor-tyrosine kinase Flt3 regulates normal hematopoiesis. An internal tandem duplication (ITD) in the juxtamembrane domain of Flt3 (Flt3-ITD) contributes to transformation and is associated with poor prognosis in acute myeloid leukemia. Here, we demonstrate that, as compared with wild-type Flt3 (Flt3-WT), Flt3-ITD more rapidly undergoes degradation through the proteasomal and lysosomal pathways in model hematopoietic 32D cells and in human leukemic MV4-11 cells. The Hsp90 inhibitor 17-allylaminodemethoxygeldanamycin (17-AAG) preferentially induced the polyubiquitination and proteasomal degradation of Flt3-ITD autophosphorylated on Tyr-591 in these cells. The E3 ubiquitin ligases c-Cbl and to a lesser extent Cbl-b facilitated at least partly Lys-48-linked polyubiquitination of autophosphorylated Flt3-ITD when coexpressed in 293T cells. Moreover, c-Cbl and Cbl-b facilitated degradation of Flt3-ITD in 293T cells and significantly enhanced the 17-AAG-induced decline in autophosphorylated Flt3-ITD. The enhancement of Flt3-ITD degradation was also observed in 32D cells inducibly overexpressing c-Cbl or Cbl-b. Furthermore, overexpression of loss-of-function mutants of both c-Cbl (c-Cbl-R420Q) and Cbl-b (Cbl-b-C373A) together in 32D cells retarded the degradation of autophosphorylated Flt3-ITD and significantly inhibited the 17-AAG-induced degradation of Flt3-ITD to confer the resistance to cytotoxicity of 17-AAG on these cells. These results suggest that c-Cbl as well as Cbl-b may play important roles in Hsp90 inhibitor-induced degradation of Flt3-ITD through the ubiquitin proteasome system and in regulation of the basal expression level of Flt3-ITD in leukemic cells.     

Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.     

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind l-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N^G-nitro-l-arginine, but not its inactive d-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.     

The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.     

Met kinase-dependent loss of the E3 ligase Cbl in gastric cancer

Strict regulation of signaling by receptor tyrosine kinases (RTKs) is essential for normal biological processes, and disruption of this regulation can lead to tumor initiation and progression. Signal duration by the Met RTK is mediated in part by the E3 ligase Cbl. Cbl is recruited to Met upon kinase activation and promotes ubiquitination, trafficking, and degradation of the receptor. The Met RTK has been demonstrated to play a role in various types of cancer. Here, we show that Met-dependent loss of Cbl protein in MET-amplified gastric cancer cell lines represents another mechanism contributing to signal dysregulation. Loss of Cbl protein is dependent on Met kinase activity and is partially rescued with a proteasome inhibitor, lactacystin. Moreover, Cbl loss not only uncouples Met from Cbl-mediated negative regulation but also releases other Cbl targets, such as the EGF receptor, from Cbl-mediated signal attenuation. Thus, Met-dependent Cbl loss may also promote cross-talk through indirect enhancement of EGF receptor signaling.     

Ubiquitination of Neuronal Nitric-oxide Synthase in the Calmodulin-binding Site Triggers Proteasomal Degradation of the Protein

Nitric-oxide synthase, a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our laboratory has discovered that certain drugs suicide-inactivate neuronal nitric-oxide synthase (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS by an Hsp70- and CHIP (C terminus of Hsc70-interacting protein)-dependent process. To further understand the process by which altered nNOS is recognized, ubiquitinated, and proteasomally degraded, we examined the sites of ubiquitination on nNOS. We utilized an in vitro ubiquitination system containing purified E1, E2 (UbcH5a), Hsp70, and CHIP that recapitulates the ability of the cells to selectively recognize and ubiquitinate the altered forms of nNOS. LC-MS/MS analysis of the tryptic peptides obtained from the in vitro ubiquitinated nNOS identified 12 ubiquitination sites. Nine of the sites were within the oxygenase domain and two were in the calmodulin-binding site, which links the oxygenase and reductase domains, and one site was in the reductase domain. Mutational analysis of the lysines in the calmodulin-binding site revealed that Lys-739 is a major site for poly-ubiquitination of nNOS in vitro and regulates, in large part, the CHIP-dependent degradation of nNOS in HEK293 cells, as well as in in vitro studies with fraction II. Elucidating the exact site of ubiquitination is an important step in understanding how chaperones recognize and trigger degradation of nNOS.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.     

The CUL7/F-box and WD Repeat Domain Containing 8 (CUL7/Fbxw8) Ubiquitin Ligase Promotes Degradation of Hematopoietic Progenitor Kinase 1

HPK1, a member of mammalian Ste20-like serine/threonine kinases, is lost in >95% pancreatic cancer through proteasome-mediated degradation. However, the mechanism of HPK1 loss has not been defined. The aims of this study are to identify the ubiquitin ligase and to examine the mechanisms that targets HPK1 degradation. We found that the CUL7/Fbxw8 ubiquitin ligase targeted HPK1 for degradation via the 26 S proteasome. The ubiquitination of HPK1 required its kinase activity and autophosphorylation. Wild-type protein phosphatase 4 (PP4), but not the phosphatase-dead PP4 mutant, PP4-RL, inhibits the interaction of Fbxw8 with HPK1 and Fbxw8-mediated ubiquitination of HPK1. In addition, we showed that Thr-355 of HPK1 is a key PP4 dephosphorylation site, through which CUL7/Fbxw8 ubiquitin ligase and PP4 regulates HPK1 stability. Knockdown of Fbxw8 restores endogenous HPK1 protein expression and inhibits cell proliferation of pancreatic cancer cells. Our study demonstrated that targeted degradation of HPK1 by the CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain the activity of HPK1 and that CUL7/Fbxw8 ubiquitin ligase promotes pancreatic cancer cell proliferation. CUL7/Fbxw8 ubiquitin ligase-mediated HPK1 degradation revealed a direct link and novel role of CUL7/Fbxw8 ubiquitin ligase in the MAPK pathway, which plays a critical role in cell proliferation and differentiation.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

Destabilization of the Epidermal Growth Factor Receptor (EGFR) by a Peptide That Inhibits EGFR Binding to Heat Shock Protein 90 and Receptor Dimerization

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.     

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF^Skp2 ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser⁶⁴ and Ser⁷², we have identified Thr⁴¹⁷ as a unique Pim-1 phosphorylation target. Phosphorylation of Thr⁴¹⁷ controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.     

Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564–574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K_d = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.     

CD81 Promotes Both the Degradation of Transferrin Receptor 2 (TfR2) and the Tfr2-mediated Maintenance of Hepcidin Expression

Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that CD81 is targeted for degradation by GRAIL, an ubiquitin E3 ligase. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6 but increased both the expression of ID1 and SMAD7, direct targets of BMP signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2 signaling pathways.     

Binding of the Receptor Tyrosine Kinase TrkB to the Neural Cell Adhesion Molecule (NCAM) Regulates Phosphorylation of NCAM and NCAM-dependent Neurite Outgrowth

Recognition molecules and neurotrophins play important roles during development and maintenance of nervous system functions. In this study, we provide evidence that the neural cell adhesion molecule (NCAM) and the neurotrophin receptor TrkB directly interact via sequences in their intracellular domains. Stimulation of TrkB by brain-derived neurotrophic factor leads to tyrosine phosphorylation of NCAM at position 734. Mutation of this tyrosine to phenylalanine completely abolishes tyrosine phosphorylation of NCAM by TrkB. Moreover, the knockdown of TrkB in hippocampal neurons leads to a reduction of NCAM-induced neurite outgrowth. Transfection of NCAM-deficient hippocampal neurons with mutated NCAM carrying an exchange of tyrosine by phenylalanine at position 734 leads to promotion of NCAM-induced neurite outgrowth in comparison with that observed after transfection with wild-type NCAM, whereas a reduction of neurite outgrowth was observed after transfection with mutated NCAM, which carries an exchange of tyrosine by glutamate that mimics the phosphorylated tyrosine. Our observations indicate a functional relationship between TrkB and NCAM.     

Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: identification of sites targeted for phosphorylation and ubiquitination

Human liver CYP2E1 is a monotopic, endoplasmic reticulum-anchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanism-based inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.     

Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.     

Ubc13 and COOH terminus of Hsp70-interacting protein (CHIP) are required for growth hormone receptor endocytosis

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(βTrCP) (Skp Cullin F-box). Despite a specific interaction between β-transducin repeat-containing protein (βTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(βTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to βTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.     

Functional Consequences of the Interactions among the Neural Cell Adhesion Molecule NCAM,the Receptor Tyrosine Kinase TrkB,and the Inwardly Rectifying K+ Channel KIR3.3

Cell adhesion molecules and neurotrophin receptors are crucial for the development and the function of the nervous system. Among downstream effectors of neurotrophin receptors and recognition molecules are ion channels. Here, we provide evidence that G protein-coupled inwardly rectifying K⁺ channel Kir3.3 directly binds to the neural cell adhesion molecule (NCAM) and neurotrophin receptor TrkB. We identified the binding sites for NCAM and TrkB at the C-terminal intracellular domain of Kir3.3. The interaction between NCAM, TrkB, and Kir3.3 was supported by immunocytochemical co-localization of Kir3.3, NCAM, and/or TrkB at the surface of hippocampal neurons. Co-expression of TrkB and Kir3.1/3.3 in Xenopus oocytes increased the K⁺ currents evoked by Kir3.1/3.3 channels. This current enhancement was reduced by the concomitant co-expression with NCAM. Both surface fluorescence measurements of microinjected oocytes and cell surface biotinylation of transfected CHO cells indicated that the cell membrane localization of Kir3.3 is regulated by TrkB and NCAM. Furthermore, the level of Kir3.3, but not of Kir3.2, at the plasma membranes was reduced in TrkB-deficient mice, supporting the notion that TrkB regulates the cell surface expression of Kir3.3. The premature expression of developmentally late appearing Kir3.1/3.3 in hippocampal neurons led to a reduction of NCAM-induced neurite outgrowth. Our observations indicate a decisive role for the neuronal K⁺ channel in regulating NCAM-dependent neurite outgrowth and attribute a physiologically meaningful role to the functional interplay of Kir3.3, NCAM, and TrkB in ontogeny.