Regulation of monoubiquitinated PCNA by DUB autocleavage

Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.     

Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases

Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed α-helical bundle through their N-terminal double GG4-like motif T⁶⁴⁸G⁶⁴⁹X₂G⁶⁵²A⁶⁵³ and glycine zipper motif T⁶⁵²X₃S⁶⁵⁶X₃G⁶⁶⁰, respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.     

Coclustering of ErbB1 and ErbB2 Revealed by FRET-Sensitized Acceptor Bleaching

Classical theory states that ligand binding induces the dimerization of ErbB proteins, leading to their activation. Although we and other investigators have shown the existence of preformed homoclusters of ErbB receptors and analyzed their composition, the stoichiometry of their heteroclusters has not been quantitatively described. Here, we report the development of the fluorescence resonance energy transfer (FRET)-sensitized acceptor bleaching (FSAB) technique to quantitate the ratio of ErbB1 and ErbB2 in their heteroclusters. In FSAB, photolabile acceptors within FRET distance from photostable donors are excited and photobleached by FRET, and the fraction of acceptors that are participating in FRET is determined. In quiescent SKBR-3 breast cancer cells, ∼35% of ErbB1 and ∼10% of ErbB2 have been found in heteroclusters. Epidermal growth factor (ligand of ErbB1) increased the fraction of ErbB2 heteroclustering with ErbB1, whereas the ratio of heteroclustered ErbB1 did not change significantly. The fractions of heteroclustered ErbB1 and ErbB2 were independent of their expression levels, indicating that the formation of these clusters is not driven by the law of mass action. In contrast, the FRET efficiency depended on the donor/acceptor ratio as expected. We present a model in which preformed receptor clusters are rearranged upon ligand stimulation, and report that the composition of these clusters can be quantitatively described by the FSAB technique.     

The Cause of ErbB2 Receptor Resistance to Downregulation

ErbB2/HER2 is a tyrosine kinase receptor belonging to the family of epidermal growth factor receptors (EGFRs); it is overexpressed in 25–30% of human breast cancer cases and has a number of structural and functional differences from other receptors of this family. Typically, the activation of tyrosine kinase receptors, i.e., formation of their homo- or heterodimers, and the subsequent signal transmission into the cell occurs when the ligand is bound to them. After dimers are formed, the internalization of a complex takes place, which plays a key role in the regulation of receptor activity. Unlike other receptors of the family, ErbB2 does not have natural ligands, but is the preferred partner for the formation of heterodimers with other members of the ErbB family. ErbB2 is also resistant to internalization and degradation. Thus, staying for a long time at the cell membrane after activation, ErbB2 continues to transmit regulatory signals to the cell nucleus. Although mechanisms ensuring the ErbB2 resistance to downregulation are not fully understood, a significant pool of experimental data suggests that such key points as interaction with Hsp90 chaperon, the ability to suppress the formation of clathrin pits, the ability to quickly return to the membrane from early endosomes, and interaction with the calcium pump PMCA2, allow ErbB2 receptor to avoid internalization.     

Regulation of Xenopus gastrulation by ErbB signaling

During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Different signals are implicated in these processes. Our previous studies reveal that signals through ErbB receptor tyrosine kinases modulate Xenopus gastrulation, but the mechanisms employed are not understood. Here we report that ErbB signals control both C&E and head mesoderm migration. Inhibition of ErbB pathway blocks elongation of dorsal marginal zone explants and activin-treated animal caps without removing mesodermal gene expression. Bipolar cell shape and cell mixing in the dorsal region are impaired. Inhibition of ErbB signaling also interferes with migration of prechordal mesoderm on fibronectin. Cell-cell and cell-matrix interaction and cell spreading are reduced when ErbB signaling is blocked. Using antisense morpholino oligonucleotides, we show that ErbB4 is involved in Xenopus gastrulation morphogenesis, and it partially regulates cell movements through modulation of cell adhesion and membrane protrusions. Our results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to non-canonical Wnt and FGF signals, that controls gastrulation. We further demonstrate that regulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation.     

Regulation of P2Y1 Receptor Traffic by Sorting Nexin 1 is Retromer Independent

The activity and traffic of G‐protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y₁ and P2Y₁₂ responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y₁ receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y₁₂ receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y₁ receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y₁ receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y₁ receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y₁ receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.     

Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements

The association of receptor tyrosine kinases is a key step in the initiation of growth factor-mediated signaling. Although the ligand-induced dimerization of inactive, monomeric receptors was the central dogma of receptor tyrosine kinase activation for decades, the existence of larger oligomers is now accepted. Both homoassociations and heteroassociations are of extreme importance in the epidermal growth factor (EGF) receptor family, leading to diverse and robust signaling. We present a statistically reliable, flow-cytometric homo-fluorescence resonance energy transfer method for the quantitative characterization of large-scale receptor clusters. We assumed that a fraction of a certain protein species is monomeric, whereas the rest are present in homoclusters of N-mers. We measured fluorescence anisotropy as a function of the saturation of fluorescent antibody binding, and fitted the model to the anisotropy data yielding the fraction of monomers and the cluster size. We found that ErbB2 formed larger homoclusters than ErbB1. Stimulation with EGF and heregulin led to a decrease in ErbB2 homocluster size, whereas ErbB1 homoclusters became larger after EGF stimulation. The activation level of ErbB2 was inversely proportional to its homocluster size. We conclude that homoclusters of ErbB1 and ErbB2 behave in a fundamentally different way. Whereas huge ErbB2 clusters serve as a reservoir of inactive coreceptors and dissociate upon stimulation, small ErbB1 homoclusters form higher-order oligomers after ligand binding.     

病毒靶向人I型干扰素受体1的负调控机制

人Ⅰ型干扰素（type I interferon, IFN-I）的诱生和应答在机体抗病毒固有免疫中发挥重要作用。但病毒多可逃逸宿主此类抗病毒免疫,导致感染和致病。Ⅰ型干扰素受体（interferon alpha receptor, IFNAR）是识别及结合IFN-I的一种跨细胞膜蛋白受体,其IFNAR1亚型在干扰素发挥抗病毒效应的启动阶段发挥关键作用;本文从IFNAR1蛋白质的表达、降解及其功能等方面,概述病毒以IFNAR1为靶点负调控IFN-I的抗病毒机制,以期为该领域基础研究和临床抗病毒策略提供有益的参考依据。     

ErbB2受体酪氨酸激酶激酶区的表达与纯化

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区.构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,选取较高水平表达菌株进行5升罐培养,以镍亲和层析手段纯化得到蛋白表达产物,进行SDS-PAGE分析和酶联免疫反应检测酶活.结果表明在毕赤酵母中成功诱导表达了约100kD的激酶融合蛋白并具有激酶活性.该研究为筛选ErbB2的抑制剂奠定了基础.     

Silencing of ErbB3/ErbB2 Signaling by Immunoglobulin-like Necl-2

ErbB2 and ErbB3, members of the EGF receptor/ErbB family, form a heterodimer upon binding of a ligand, inducing the activation of Rac small G protein and Akt protein kinase for cell movement and survival, respectively. The enhanced ErbB3/ErbB2 signaling causes tumorigenesis, invasion, and metastasis. We found here that the ErbB3/ErbB2 signaling is regulated by immunoglobulin-like Necl-2, which is down-regulated in various cancer cells and serves as a tumor suppressor. The extracellular region of ErbB3, but not ErbB2, interacted in cis with that of Necl-2. This interaction reduced the ligand-induced, ErbB2-catalyzed tyrosine phosphorylation of ErbB3 and inhibited the consequent ErbB3-mediated activation of Rac and Akt, resulting in the inhibition of cancer cell movement and survival. These inhibitory effects of Necl-2 were mediated by the protein-tyrosine phosphatase PTPN13 which interacted with the cytoplasmic tail of Necl-2. We describe here this novel mechanism for silencing of the ErbB3/ErbB2 signaling by Necl-2.ErbB2 and ErbB3 are members of the EGF receptor/ErbB family, which has ErbB1 and ErbB4 as additional members (1). ErbB2 and ErbB3 are also known as HER2/Neu and HER3, respectively. No ligands binding directly to ErbB2 have been identified yet, whereas heregulin (HRG)³-α and -β, also known as neuregulin-1 and -2, respectively, directly bind to ErbB3. ErbB2 and ErbB3 have kinase domains in their cytoplasmic tails, but that of ErbB3 lacks kinase activity. Therefore, the homodimer of ErbB3 formed by binding of HRG does not transduce any intracellular signaling. By contrast, ErbB2 heterophilically interacts in cis with HRG-occupied ErbB3 and phosphorylates nine tyrosine residues of ErbB3, causing recruitment and activation of the p85 subunit of phosphoinositide 3-kinase (PI3K) and the subsequent activation of Rac small G protein and Akt protein kinase (2). The activation of Rac enhances cell movement and that of Akt prevents cell apoptosis (3).ErbB2 serves as an oncogenic protein (4), and amplification of the ErbB2 gene is observed in many types of cancers. For instance, it is amplified in ∼3% of lung cancers, ∼30% of breast cancers, ∼20% of gastric cancers, and ∼60% of ovarian cancers (5). Moreover, mutation of the ErbB2 gene is found in many types of cancers, namely, ∼10% of lung cancers, ∼4% of breast cancers, ∼5% of gastric cancers, and ∼3% of colorectal cancers (6). This gene amplification or mutation causes enhanced signaling for cell movement and survival, eventually resulting in tumorigenesis, invasiveness, and metastasis. On the basis of these properties of ErbB2, it has been recognized as a good target for cancer therapy; indeed, ErbB2-targeting drugs have already been developed and used clinically (7, 8). However, it remains unknown whether ErbB2 is involved in oncogenesis in cancers in which its gene is not amplified or mutated. In addition, it was recently reported that overexpression of ErbB3 is also involved in tumor malignancy (9), but it remains unknown how ErbB3 serves as an oncogenic protein in cancers in which it is not overexpressed.The nectin-like molecule (Necl) family consists of five members, Necl-1, Necl-2, Necl-3, Necl-4, and Necl-5, and comprises a superfamily with the nectin family, which consists of four members, nectin-1, nectin-2, nectin-3, and nectin-4 (10). All members of this superfamily have similar domain structures: they have one extracellular region with three Ig-like loops, one transmembrane segment, and one cytoplasmic tail. We recently found that the extracellular region of Necl-5 directly interacts in cis with that of the platelet-derived growth factor (PDGF) receptor and that this interaction enhances the PDGF-induced cell proliferation and movement (11–14). Necl-5 is up-regulated in many types of cancer cells and causes at least partly enhanced movement and proliferation of cancer cells (11, 12). These earlier findings prompted us to study the potential interaction of other Necls with other growth factor receptors. Consequently, we found here that the extracellular region of Necl-2 directly interacts in cis with that of ErbB3, but not ErbB2, and reduces the HRG-induced signaling pathways of the ErbB3/ErbB2 heterodimer for cell movement and survival.Necl-2 is known by many names: IgSF4a, RA175, SgIGSF, TSLC1, and SynCAM1 (15–19). Necl-2 was directly reported in GenBank^TM in 1998; IgSF4a was identified as a candidate for a tumor suppressor gene in the loss of heterozygosity region of chromosome 11q23.2 (16); RA175 was identified as a gene highly expressed during the neuronal differentiation of embryonic carcinoma cells (19); SgIGSF was identified as a gene expressed in spermatogenic cells during the early stages of spermatogenesis (18); TSLC1 was identified as a tumor suppressor in human non-small cell lung cancer (17); and SynCAM1 was identified as a brain-specific synaptic adhesion molecule (15). In this study, we use the name “Necl-2,” because it was first reported.Necl-2 shows Ca²⁺-independent homophilic cell-cell adhesion activity and Ca²⁺-independent heterophilic cell-cell adhesion activity with other members of the nectin and Necl families, Necl-1 and nectin-3, and another Ig-like molecule with two Ig-like loops, CRTAM (20–22). These cell-cell adhesion activities are mediated by their extracellular regions. Necl-2 is associated with many peripheral membrane proteins through its cytoplasmic tail. The juxtamembrane region of the cytoplasmic tail contains a band 4.1-binding motif and binds the tumor suppressor DAL-1, a band 4.1 family member, which connects Necl-2 to the actin cytoskeleton (23). In addition, the cytoplasmic tail contains a PDZ domain-binding motif in its C-terminal region and binds Pals2, Dlg3/MPP3, and CASK, which are MAGUK subfamily members having an L27 domain (15, 20, 24, 25). However, the exact roles of the binding of these molecules to Necl-2 remain unknown.Necl-2 is widely expressed in various tissues and organs, and abundantly expressed in epithelial cells (20, 26). Its expression is down-regulated in many types of cancer cells owing to hypermethylation of the Necl-2 gene promoter and/or loss of heterozygosity of 11q23.2 (26). Its expression is also undetectable in fibroblasts, such as NIH3T3, Swiss3T3, and L cells (20). Necl-2 has been shown to be a tumor suppressor in human non-small cell lung cancer (17), but it remains unknown how it fulfills this role. The relationship between Necl-2 and the ErbB family remains unknown, either. In addition, the heterophilic interaction of Necl-2 with CRTAM enhances the cytotoxicity of NK cells and the secretion of γ-interferon from CD8⁺ T cells to attack the Necl-2-expressing cells (22, 27). Studies using Necl-2-deficient mice have revealed that Necl-2 in Sertoli cells is an important cell adhesion molecule for Sertoli-spermatid junctions during spermatogenesis (28–30). In the seminiferous tubules of Necl-2-deficient mice, round and elongating spermatids with a distorted shape are generated owing to failure of contact with Sertoli cells, resulting in male-specific infertility. In the present study, we focused on the role of Necl-2 as a tumor suppressor and clarified its mode of action.     

ErbB2 and ErbB4 Cbl binding sites can functionally replace the ErbB1 Cbl binding site

Poor downregulation of ErbB receptors is associated with enhanced downstream signaling and tumorigenesis. It has been suggested that poor downregulation of ErbB-2, -3 and -4 receptors when compared to ErbB1 is due to decreased recruitment of Cbl E3 ligase proteins. However, a highly conserved Cbl binding site is not only present in ErbB1/EGFR (FLQRpY¹⁰⁴⁵SSDP), but also in ErbB2 (PLQRpY¹⁰⁹¹SEDP) and ErbB4 (STQRpY¹¹⁰³SADP). We therefore replaced the ErbB1 Cbl binding site by that of ErbB2 and ErbB4. Whereas retrovirally infected NIH3T3 cells containing the EGFR Y1045F mutation showed dramatically impaired Cbl recruitment, EGFR ubiquitination and delayed EGFR degradation, replacement of the EGFR Cbl binding site by that of ErbB2 or ErbB4 did not affect Cbl recruitment, receptor-ubiquitination, -degradation, -downregulation or ligand degradation. We conclude that poor downregulation of ErbB2 and ErbB4 receptors is not due to sequence variations in the Cbl binding site of these receptors.     

Modulation of ErbB2 Blockade in ErbB2-Positive Cancers: The Role of ErbB2 Mutations and PHLDA1

We set out to study the key effectors of resistance and sensitivity to ErbB2 tyrosine kinase inhibitors, such as lapatinib in ErbB2-positive breast and lung cancers. A cell-based in vitro site-directed mutagenesis lapatinib resistance model identified several mutations, including the gatekeeper ErbB2 mutation ErbB2-T798I, as mediating resistance. ErbB2-T798I engineered cell models indeed show resistance to lapatinib but remain sensitive to the irreversible EGFR/ErbB2 inhibitor, PD168393, suggestive of potential alternative treatment strategies to overcome resistance. Gene expression profiling studies identified a select group of downstream targets regulated by ErbB2 signaling and define PHLDA1 as an immediately downregulated gene upon oncogenic ErbB2 signaling inhibition. We find significant down-regulation of PHLDA1 in primary breast cancer and PHLDA1 is statistically significantly less expressed in ErbB2 negative compared with ErbB2 positive tumors consistent with its regulation by ErbB2. Lastly, PHLDA1 overexpression blocks AKT signaling, inhibits cell growth and enhances lapatinib sensitivity further supporting an important negative growth regulator function. Our findings suggest that PHLDA1 might have key inhibitory functions in ErbB2 driven lung and breast cancer cells and a better understanding of its functions might point at novel therapeutic options. In summary, our studies define novel ways of modulating sensitivity and resistance to ErbB2 inhibition in ErbB2-dependent cancers.     

Production of the Extracellular Part of the ErbB2 Receptor for the Study of Immunobiologicals

Russian Journal of Bioorganic Chemistry - The extracellular part of the ErbB2 receptor (ecdErbB2), an oncological marker and a target for therapeutic antibodies, has been expressed in eukaryotic...     

癌基因MDM2对雌激素受体转录活性的调节作用

目的:检测MDM2基因对雌激素受体α和β(ERα和ERβ)是否具有转录活性调节作用。方法:用PCR方法从乳腺文库中扩增MDM2序列,并将其以正确相位与pcDNA3-FLAG载体中的FLAG序列融合,构建成重组质粒pcDNA3-FLAG-MDM2;以含雌激素反应元件的荧光素酶(ERE-LUC)为报告基因,通过检测荧光素酶活性来确定MDM2是否对ER有转录调节因子的作用。结果:克隆和表达了MDM2基因;MDM2只对ERα具有转录活性调节作用。结论:MDM2对ER转录活性的调节具有亚型特异性。     

Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+-release Channel RyR1 by S-Palmitoylation

The ryanodine receptor/Ca²⁺-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca²⁺ release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca²⁺ channel Ca_V1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in “hot spot” regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca²⁺ release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca²⁺-ATPase 1A and the α_1S subunit of the L-type Ca²⁺ channel Ca_V1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca²⁺ flux in skeletal muscle that mediates excitation-contraction coupling.