Tyrosine phosphorylation of protein kinase D2 mediates ligand-inducible elimination of the Type 1 interferon receptor

Type 1 interferons (including IFNα/β) activate their cell surface receptor to induce the intracellular signal transduction pathways that play an important role in host defenses against infectious agents and tumors. The extent of cellular responses to IFNα is limited by several important mechanisms including the ligand-stimulated and specific serine phosphorylation-dependent degradation of the IFNAR1 chain of Type 1 IFN receptor. Previous studies revealed that acceleration of IFNAR1 degradation upon IFN stimulation requires activities of tyrosine kinase TYK2 and serine/threonine protein kinase D2 (PKD2), whose recruitment to IFNAR1 is also induced by the ligand. Here we report that activation of PKD2 by IFNα (but not its recruitment to the receptor) depends on TYK2 catalytic activity. PKD2 undergoes IFNα-inducible tyrosine phosphorylation on specific phospho-acceptor site (Tyr-438) within the plekstrin homology domain. Activated TYK2 is capable of facilitating this phosphorylation in vitro. Tyrosine phosphorylation of PKD2 is required for IFNα-stimulated activation of this kinase as well as for efficient serine phosphorylation and degradation of IFNAR1 and ensuing restriction of the extent of cellular responses to IFNα.     

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.     

Polyubiquitination of insulin-like growth factor I receptor (IGF-IR) activation loop promotes antibody-induced receptor internalization and down-regulation

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

Role of p38 protein kinase in the ligand-independent ubiquitination and down-regulation of the IFNAR1 chain of type I interferon receptor

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of type I interferon (IFN) receptor is a robust and specific mechanism that limits the magnitude and duration of IFNα/β signaling. Besides the ligand-inducible IFNAR1 degradation, the existence of an "inside-out" signaling that accelerates IFNAR1 turnover in the cells undergoing the endoplasmic reticulum (ER) stress and activated unfolded protein responses has been recently described. The latter pathway does not require either presence of ligands (IFNα/β) or catalytic activity of Janus kinases (JAK). Instead, this pathway relies on activation of the PKR-like ER kinase (PERK) and ensuing specific priming phosphorylation of IFNAR1. Here, we describe studies that identify the stress activated p38 protein kinase as an important regulator of IFNAR1 that acts downstream of PERK. Results of the experiments using pharmacologic p38 kinase inhibitors, RNA interference approach, and cells from p38α knock-out mice suggest that p38 kinase activity is required for priming phosphorylation of IFNAR1 in cells undergoing unfolded protein response. We further demonstrate an important role of p38 kinase in the ligand-independent stimulation of IFNAR1 ubiquitination and degradation and ensuing attenuation of IFNα/β signaling and anti-viral defenses. We discuss the distinct importance of p38 kinase in regulating the overall responses to type I IFN in cells that have been already exposed to IFNα/β versus those cells that are yet to encounter these cytokines.     

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.     

A new highly efficient substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B) reveals full autoactivation of the insulin receptor precursor

PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.     

Involvement of protein kinase Cdelta/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 (PARP-1) signaling pathway in histamine-induced up-regulation of histamine H1 receptor gene expression in HeLa cells

The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.     

Protein kinase R as mediator of the effects of interferon (IFN) gamma and tumor necrosis factor (TNF) alpha on normal and dysplastic hematopoiesis

IFNγ and TNFα are potent inhibitors of hematopoiesis and have been implicated in the pathophysiology of bone marrow failure and myelodysplastic syndromes (MDS). We examined the role of protein kinase R (PKR) in the generation of the inhibitory effects of these myelosuppressive cytokines on hematopoiesis. Our data demonstrate that PKR is rapidly phosphorylated/activated in response to engagement of IFNγ or TNFα receptors in normal human hematopoietic progenitors. Such engagement of PKR is important for the suppressive effects of these cytokines on normal hematopoiesis. Pharmacological targeting of PKR using a specific inhibitor or siRNA-mediated PKR knockdown results in partial reversal of the suppressive effects of IFNγ and TNFα on normal human CD34+-derived myeloid (colony-forming unit-granulocyte-monocytic) and erythroid (burst-forming unit-erythroid) progenitors. Importantly, inhibition of PKR activity or expression increases hematopoietic colony formation from human MDS progenitors, suggesting that drugs that target PKR may provide a novel approach for the treatment of MDS and marrow failure syndromes. Altogether, our data establish that beyond its key role in the induction of IFN-antiviral responses, PKR plays important roles in signaling for IFNγ and other myelosuppressive cytokine receptors as a common mediator of signals for hematopoietic suppression.     

Down-regulation of CMTM8 induces epithelial-to-mesenchymal transition-like changes via c-MET/extracellular signal-regulated kinase (ERK) signaling

The acquisition of an invasive phenotype is a critical turning point for malignant tumor cells. CMTM8, a potential tumor suppressor, is frequently down-regulated in solid tumors, and its overexpression induces tumor cell apoptosis. Here, we identify a new role for CMTM8 in regulating tumor cell migration. Reducing CMTM8 expression in HepG2 hepatocellular carcinoma cells results in the acquisition of epithelial-to-mesenchymal transition (EMT) features, including a morphological change from organized epithelial sheets to scattered fibroblast-like shapes, reduction of the epithelial marker E-cadherin, and an increased invasive and migratory ability. These phenotypic changes are mediated in large part by the ERK-MAPK pathway, as the MEK inhibitor U0126 and shRNA-mediated knockdown of ERK2 significantly reversed these phenotypes. Hepatocyte growth factor binding to the c-MET receptor is known to induce EMT in HepG2 cells. We found that CMTM8 knockdown in HepG2 cells induced c-MET signaling and ERK activation. Inhibition of c-MET signaling with the small molecule inhibitor SU11274 or c-MET RNAi blocked the EMT-like changes following CMTM8 knockdown. CMTM8 overexpression in HepG2 cells inhibited hepatocyte growth factor-induced EMT-like morphological changes and cell motility. Down-regulation of CMTM8 also promoted an EMT-like change in MCF-10A cells, indicating a broader role for CMTM8 in regulating cellular transformation.     

Inhibition of RNase L and RNA-dependent protein kinase (PKR) by sunitinib impairs antiviral innate immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.     

Phosphorylation of G protein-coupled receptor kinase 1 (GRK1) is regulated by light but independent of phototransduction in rod photoreceptors

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser(21) in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark- and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser(21). Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin α-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.     

TBK1-associated protein in endolysosomes (TAPE) is an innate immune regulator modulating the TLR3 and TLR4 signaling pathways

The innate immune system elicits the first wave of immune responses against pathogen infection. Its operational modes are complex and have yet to be defined. Here, we report the identification of an innate immune regulator termed TAPE (TBK1-associated protein in endolysosomes), previously known as CC2D1A/Freud-1/Aki-1, which modulates the TLR3 and TLR4 pathways. We found that TAPE activated the TBK1, NF-κB, and ERK pathways leading to IFN-β and inflammatory cytokine induction. TAPE was shown to colocalize with endosomal marker Rab5 and lysosomal marker LAMP1 in mammalian cells, suggesting that TAPE resided in endolysosomes. Knockdown of TAPE selectively impaired the TLR3 and endocytic TLR4 pathways to IFN-β induction. Furthermore, TAPE interacted and synergized with Trif to activate IFN-β. TAPE knockdown failed to block Trif-mediated IFN-β induction, whereas Trif knockdown impaired the TLR3 and TAPE cooperation on IFN-β induction, suggesting that TAPE acts upstream of Trif. Together, our data demonstrate a central role for TAPE in linking TLR3 and TLR4 to innate immune defenses at an early step.