Interleukin-4-receptor signal transduction [Elektronische Ressource] : involvement of P62 / vorgelegt von Susanne Bürgis

Interleukin-4 ReceptorSignal Transduction:Involvement of P62Den Naturwissenschaftlichen Fakult¨atender Friedrich–Alexander–Universitat Erlangen–Nurnberg¨ ¨zurErlangung des Doktorgradesvorgelegt vonSusanne Bu¨rgisaus Nurnberg¨Als Dissertation genehmigt von denNaturwissenschaftlichen Fakult¨atender Universitat Erlangen–Nurnberg¨ ¨Tag der mu¨ndlichen Pru¨fung: 6. Oktober 2006Vorsitzender derPrufungskommission: Prof. Dr. D.–P. Hader¨ ¨Erstberichterstatter: Prof. Dr. Dr. A. GessnerZweitberichterstatter: Prof. Dr. T. WinklerContents1 Introduction 11.1 The biology of interleukin-4 and its receptor . . . . . . . . . . . . . . . . . 11.1.1 Interleukin-4 and its biological functions . . . . . . . . . . . . . . . 11.1.2 Interleukin-4-receptor complexes . . . . . . . . . . . . . . . . . . . . 21.1.3 Interleukin-4-receptor signaling . . . . . . . . . . . . . . . . . . . . 31.2 The adaptor protein P62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.3 Link between IL-4R and P62 . . . . . . . . . . . . . . . . . . . . . . . . . . 71.4 Aims of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Materials and Methods 112.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1.1 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1.2 Commercial systems . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . .
Publié le : dimanche 1 janvier 2006
Lecture(s) : 34
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2006/461/PDF/SUSANNEB%FCRGISDISSERTATION.PDF
Nombre de pages : 83
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Interleukin-4 Receptor Signal Transduction: Involvement of P62
DenNaturwissenschaftlichenFakultaten derFriedrichAlexanderUniversitatErlangenNurnberg zur Erlangung des Doktorgrades
vorgelegt von SusanneBurgis s N berg au urn
Als Dissertation genehmigt von den NaturwissenschaftlichenFakultaten derUniversitatErlangenNurnberg
TagdermundlichenPrufung:
Vorsitzender der Prufungskommission: Erstberichterstatter: Zweitberichterstatter:
6. Oktober 2006
Prof.Dr.D.P.Hader Prof. Dr. Dr. A. Gessner Prof. Dr. T. Winkler
Contents
1 Introduction 1.1 The biology of interleukin-4 and its receptor . . . . . . . . . . . . . . . . . 1.1.1 Interleukin-4 and its biological functions . . . . . . . . . . . . . . . 1.1.2 Interleukin-4-receptor complexes . . . . . . . . . . . . . . . . . . . . 1.1.3 Interleukin-4-receptor signaling . . . . . . . . . . . . . . . . . . . . 1.2 The adaptor protein P62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Link between IL-4R and P62 . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Aims of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials and Methods 2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Commercial systems . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.5 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.6 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.7 Cell lines and bacteria . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.8 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.9 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Genotyping of mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Polymerase chain reaction . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Sequencing of plasmids and PCR products . . . . . . . . . . . . . . 2.2.4 Isolation of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . 2.2.5 Southern blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.6 Isolation of total RNA . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.7 Reverse transcription RT–PCR . . . . . . . . . . . . . . . . . . . . 2.2.8 Quantitative RT–PCR . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.9 Enzyme linked immuno sorbent assay . . . . . . . . . . . . . . . . . 2.2.10 Generation of a P62 antiserum . . . . . . . . . . . . . . . . . . . . . 2.2.11 Transfection of cells with calcium–phosphate . . . . . . . . . . . . . 2.2.12 Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.13 Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.14 Proliferation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.15 Stimulation of macrophages . . . . . . . . . . . . . . . . . . . . . .
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1 1 1 2 3 5 7 9 11 11 11 11 12 12 13 14 16 17 17 18 18 18 19 19 20 20 20 20 21 21 22 22 22 23 23
iv CONTENTS 2.2.16 Manipulation of mouse embryonic stem cells . . . . . . . . . . . . . 24 2.2.17 Preparation of mouse embryo fibroblasts . . . . . . . . . . . . . . . 24 2.2.18 Class switching of splenic B cells . . . . . . . . . . . . . . . . . . . 25 2.2.19TH2dierentiationofnaıveTcells..................25 2.2.20 Infection withLeishmania major. . . . . . . . 26. . . . . . . . . . . 2.2.21 Oral glucose tolerance of mice . . . . . . . . . . . . . . . . . . . . . 26 2.2.22 OVA induced allergic lung disease . . . . . . . . . . . . . . . . . . . 26 3 Results 27 3.1 Studies in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.1.1 Phosphorylation of signaling intermediates . . . . . . . . . . . . . . 27 3.1.2 Effect of kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . 27 3.2 Involvement of P62 in IL-4R signaling . . . . . . . . . . . . . . . . . . . . . 30 3.2.1 Binding of P62 to the IL-4R . . . . . . . . . . . . . . . . . . . . . . 30 3.2.1.1 Colocalization of P62-EGFP and IL-4R-RFP . . . . . . . 30 3.2.1.2 Co-Immunoprecipitation of P62 with the IL-4R . . . . . . 30 3.2.1.3 Trimolecular Co-Immunoprecipitation . . . . . . . . . . . 31 3.2.2 Description of the P62 gene deficient mouse model . . . . . . . . . . 32 3.2.2.1 P62 deficiency on genomic level . . . . . . . . . . . . . . . 32 3.2.2.2 P62 deficiency on transcriptome level . . . . . . . . . . . . 32 3.2.2.3 P62 deficiency on protein level . . . . . . . . . . . . . . . 34 3.2.3 Steady state phenotype of P62 gene-targeted mice . . . . . . . . . . 35 3.2.4 Immunological phenotypes of P62 gene-targeted mice . . . . . . . . 36 3.2.4.1 Oral glucose tolerance . . . . . . . . . . . . . . . . . . . . 36 3.2.4.2 Functionality of macrophages . . . . . . . . . . . . . . . . 37 3.2.4.3 Class switching of splenic B cellsin vitro. . . . . . . . . . 38 3.2.4.4 Differentiation of naıve T cellsin vitro. . . . . . . . . . . 39 3.2.4.5 Infection withLeishmania major. . . . . . . . . . . . . . 41 3.2.4.6 Fibroblast functionality . . . . . . . . . . . . . . . . . . . 42 3.2.4.7 OVA induced allergic asthma . . . . . . . . . . . . . . . . 43 3.3 Generation of a ΔN388 IL-4R mouse model . . . . . . . . . . . . . . . . . 44 3.3.1 Targeting strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.2 Homologous recombination . . . . . . . . . . . . . . . . . . . . . . . 45 3.3.3 Blastocyst injection and breeding strategy . . . . . . . . . . . . . . 46 4 Discussion 47 4.1 Mechanism of IL-4 induced proliferation in TF1 cells . . . . . . . . . . . . 47 4.2 Binding of P62 to IL-4R and PKCζ 49. . . . . . . . . .. . . . . . . . . . . . 4.3 Involvement of P62 in IL-4R signal transduction . . . . . . . . . . . . . . . 50 4.4 Generation of a ΔN388 IL-4R knock in mouse model . . . . . . . . . . . . 52 5 Summary 55 A Abbreviations 59 Bibliography 61
Chapter 1
Introduction
1.1 The biology of interleukin-4 and its receptor
1.1.1 Interleukin-4 and its biological functions Murine interleukin-4 (IL-4) is a pleiotropic cytokine of 140 amino acids that is secreted after removal of a signal peptide as a mature protein of 120 amino acids, and has first been described as a growth factor for B cells by the group of William Paul [58]. Similar to other cytokines, it has a compact, globular fold, which is stabilized by three disulphide bonds [18]. The crystal structure has been solved for the human protein, and based on the amino acid sequence identity, the murine protein is expected to have a similar tertiary structure dominated by a four alpha-helix bundle with a left handed twist [153]. IL-4 is mainly sectreted by CD4+T cells that also produce IL-5 and IL-13 (TH2 cells) [128]. In mice, basophils have been shown to secrete IL-4 after infection with the nematode Nippostrongylus brasiliensisin humans upon challenge with allergens [127]; likewise,[93], mast cells have been reported to secrete IL-4 [13, 15]. Additional sources of IL-4 are NK1.1 positive CD4+T cells [20],γ/δ also Recently,T cells [35] and eosinophils [31]. dendritic cells and B cells have been suggested to produce IL-4 [90, 52]. IL-4 is an important cytokine in shaping immune responses and exerts different effects on various hematopoietic cells including lymphocytes (reviewed in [43] and [101]). On B cells, it acts as a growth factor [58] and is responsible for the upregulation of surface molecules such as MHC II [103], the low affinity IgE receptor CD23 [25] and the IL-4R itself [104]. Likewise, IL-4 induces Ig heavy chain class switching of human B cells to IgE and IgG4[39] and is indispensable for class switching of murine B cells to IgE and IgG1 [23, 150]. On T cells, IL-4 acts as a growth factor that induces either proliferation or antiapoptotic effects [60]. IL-4 is also required for the development of TH2 cells from naıve T cells after antigen stimulation. These cells secrete large amounts of IL-4 and other cytokines such as IL-5 or IL-13, thereby initiating a positive feedback loop. Additionally, IL-4 blocks the development of IFN-γsecreting TH1 cells, thus stabilizing a TH2 dominated immune response [59, 128]. In some infectious disease models it is decisive for the outcome of the infection, whether the immune system mounts a TH1- or TH2-dominated immune response. The importance of IL-4 for this decision has been clearly shown in various mouse
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CHAPTER 1. INTRODUCTION
models using either neutralizing anti-IL-4 antibodies [117, 121], soluble IL-4R [113, 44] or IL-4 gene-deficient mice [75]. Apart from lymphocytes, IL-4 also affects other cells of the hematopoietic lineage. It upregulates MHC II expression on monocytes and leads to enhanced antigen presentation by macrophages, while at the same time the production of proinflammatory cytokines is downregulated [10]. IL-4 is also involved in inflammatory processes. It influences the expression of endothelial adhesion molecules like vascular cell adhesion molecule 1 (VCAM-1) [145] or E-selectin [6] and promotes chemokine production, thereby favouring the recruitment of eosinophils to the site of infection. Dysregulation of IL-4 expression results in uncontrolled allergic inflammation in mice and humans [115, 143], so a thorough understanding of IL-4R signal transduction processes is essential for therapeutic applications.
1.1.2 Interleukin-4-receptor complexes IL-4 binds to a high affinity receptor IL-4Rα(Kd20 to 80 pM [87]), that is a member of the type I cytokine receptor family as it contains conserved cysteine residues and an extracellular WSXWS motif, which is required for ligand binding [85]. The receptor is ex-pressed on a variety of hematopoietic (monocytes, macrophages, mast cells, T cells and B cells) and nonhematopoietic cells (fibroblasts, neuroblasts, keratinocytes and hepatocytes) at relatively low frequencies (100 to 5000 copies per cell). Apart from the glycosylated 140 kD transmembrane protein, a 40 kD soluble IL-4R has been identified, that results from an alternatively spliced mRNA [99]. Compared to the transmembrane receptor, the soluble IL-4R binds IL-4 with similar affinity [34] and has been employed in the treatment of human asthma [11] and murine leishmaniasis [44]. Signal tranduction can be initiated through artificial cross-linkage of IL-4Rαchains [80], whereas physiological activity is obtained through heterodimerization of the IL-4Rαchain with either the common gamma chain (γcto yield the type I receptorchain, CD132) complex, or the IL-13Rα The1 chain to yield the type II receptor complex, respectively. majority of cells, including hematopoietic cells, express the type I IL-4R complex in which γc protein is also involved in the signal transduction of receptor Thischain participates. complexes for IL-2, IL-7, IL-9, IL-15 [138] and IL-21 [108]. First, a complex of IL-4 and IL-4Rαis formed and subsequentlyγcchain interacts with this complex [82]; this binding does not increase the affinity for IL-4, but is required for the initiation of signal transduc-tion [119]. Cells lacking theγcchain (mostly nonhematopoietic cells) can signal via the type II IL-4R complex [16, 56]. This receptor can also transduce signals in response to the cytokine IL-13 [92]. Additionally to the IL-13Rα1 chain, an IL-13Rα2 chain exists, that binds IL-13 with even higher affinity but does not contribute to signal transduction; it acts as a decoy receptor for IL-13 [158]. However, most recently the IL-13Rα2 chain was described to transduce IL-13 signals in macrophages, thereby fostering TGF-β-mediated fibrosis [36]. Recently the IL-4Rαchain has been proposed to contribute to the signal transduction of progesterone-induced blocking factor (PIBF), which induces a TH2 dominated cytokine
1.1. THE BIOLOGY OF INTERLEUKIN-4 AND ITS RECEPTOR 3
production through signaling via STAT6. This novel receptor complex consists of the IL-4Rαchain and the GPI-anchored PIBF receptor [77].
1.1.3 Interleukin-4-receptor signaling As a typical member of the hematopoietin receptor family, the IL-4Rαand the associ-ated receptor moleculesγcchain and IL-13Rα1 lack endogenous kinase activity. Thus, for the initiation of signaling cascades, intracellular kinases and adaptor proteins have to be recruited. Janus tyrosine kinases (Jaks) are known to contribute to cytokine signaling [62]: Jak-1, Jak-2 and Jak-3 have been shown to interact with the IL-4R. The IL-4Rα chain itself recruits Jak-1 and in some cell lines also Jak-2 [101]; in doing so, binding is mediated by a conserved motif of the IL-4Rαchain, located in close proximity to the transmembrane region and containing several acidic amino acids (box-1 motif) [27, 95]. Theγcand has been shown to bind Jak-3, whereas thechain also contains a box-1 motif IL-13Rα1 chain is associated with Jak-2 or the tyrosine kinase 2 (Tyk2) [106]. Activation of Jaks or other IL-4R associated kinases leads to tyrosine phosphorylation of the IL-4Rαconserved tyrosine residues of the intracellular receptor Five chain [130]. portion are phosphorylated, namely tyrosines Y475, Y550, Y578, Y606 and Y684 (num-bering according to Mosleyet al. adaptor proteins with Src homology[99]). Subsequently, 2 domains (SH2) or phosphotyrosine-binding domains (PTB) can bind to the receptor. Two major pathways for IL-4R signal transduction have been described [101]: one em-ploys insuline receptor substrates (IRS) and transduces mainly IL-4 induced proliferation signals (IRS-pathway), the other uses signal transducers and activators of transcription (STATs) and mediates gene activation in response to IL-4 (Jak/STAT-pathway).
Figure 1.1:type I and type II. Schematic representation with receptor chains involvedIL-4R and conserved interaction domains for adaptor proteins.
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CHAPTER 1. INTRODUCTION
Signaling via the IRS-pathway is initiated by the recruitment of IRS molecules, that possess a PTB domain, to the phosphorylated consensus motif466PIxxxxNPxYxSxSD480 within the IL-4Rαchain [71] (see figure 1.1). motif is also known as insulin-/IL-4R- This motif (I4R) due to the homology in receptor sequences [155]. Subsequently, IRS molecules are multiply phosphorylated at up to 20 different amino acids by Jaks [140, 160], after which they act as adaptor proteins linking other signal transduction molecules to the re-ceptor [139, 141]. Phosphorylated IRS molecules have been described to bind to the reg-ulatory subunit of the phosphoinositide-3 kinase (PI3-kinase); the activated PI3-kinase phosphorylates membrane lipids that act as second messengers and are crucial for the survival of the cell [37]. Another binding partner of phosphorylated IRS is the adaptor molecule Grb-2, that establishes a link to the Ras/Raf-pathway [26]. Similarly, signaling via the STAT6-pathway requires the recruitment of cytoplasmatic STAT6 molecules to the IL-4Rα three conserved tyrosine residues Y550, Y578chain. The and Y606 of the IL-4Rαact as binding sites for the SH2 domain of STAT6 (see figure 1.1), and bound STAT6 is phosphorylated by Jaks. After tyrosine phosphorylation, STAT6 dis-sociates from the receptor and translocates as a homodimer to the nucleus, where it acts as a transcription factor binding to promotor consensus motifs (GAS-elements) of IL-4 inducible genes [63, 91]. STAT6 regulates genes responsible for allergic reactions, a TH2-dominated immune response and IgE production [110].
Figure 1.2:IL-4R deletion mutants. Schematic representation of C-terminal IL-4Rαdele-tion mutants and their ability to proliferate upon IL-4 stimulation and to activate signaling intermediates.
1.2. THE ADAPTOR PROTEIN P62
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However, identification of functional regions within IL-4Rαbeing strictly responsible for either proliferation or gene activation is difficult. On the one hand, the IRS-pathway also phosphorylates high mobility group protein-I (HMG-I), a DNA-binding protein involved in the regulation of Iǫexpression in response to IL-4 [154], hence influencing gene ex-pression (IgE class switching). Also, cell lines transfected with truncated human IL-4Rα constructs, that lack all STAT6 binding sites, were still able to signal weak IL-4 induced gene activation as measured by surface expression of CD23 [120]. More recently, an acidic serine rich motif of the IL-4Rαtermed ID-1 (347EDVKEMEDNSPEMSLSGEEEVSQV370) has been connected to IL-4 induced activation of STAT6 and also STAT5 [97, 136], which resulted in the induction of TH2 differentiation in activated T cells. On the other hand, activated STAT6 also leads to the expression of genes important for survival and prolifer-ation of lymphocytes like growth factor-independent gene-1 (GFI-1) or E4 binding protein 4 (E4BP4) [162]. Data from our own laboratory also challenge the model of IL-4R signal transduction de-scribed above: using a cell culture system of human TF-1 cells, that were stably transfected with truncated murine IL-4Rαconstructs, it was shown that IL-4 induced proliferation required neither phosphorylation of IRS-2 nor STAT6 (see figure 1.2), whereas phospho-rylation of Jak1 was a strict prerequisite for functionality [144]. As functional receptors lacked binding sites for both IRS molecules and STAT6, and alternative activation of these adaptor proteins was not observed, the existence of an alternative signal transduc-tion pathway for IL-4 signals, employing additional adaptor proteins, was postulated.
1.2 The adaptor protein P62 In the literature P62 protein has been described in a variety of different contexts and under various synonyms: in humans, the protein has been identified as a phosphotyrosine-independent ligand of the Src homology 2 (SH2) domain of p56lck[68, 107]; in rat, the protein was shown to interact with the regulatory domain of the atypical protein kinase C-ζ[114], and was therefore termed PKCζinteracting protein (ZIP). In the murine system, P62 has been described as an oxidative stress protein induced in peritoneal macrophages (A170) [64], and also as signal transduction and adaptor protein (STAP) expressed in osteoblasts [105]. Amino acid sequences are highly conserved between species, the murine protein shares 97% and 90% homology with rat and human P62, respectively. Proteins share several structural motifs, including an SH2 binding domain, an acidic interaction domain (AID) that binds the atypical PKCζ, a ZZ zink finger, a binding site for TNF receptor associated factor 6 (TRAF6), two PEST sequences that are associated with the rapid degradation of proteins, and a ubiquitin associated (UBA) domain (see figure 1.3) [40]. Two splice variants of rat P62, termed ZIP2 and ZIP3 have been identified, that lack the TRAF6 binding domain or the PEST and UBA domains, respectively [24, 46]. Endogenous P62 is located either in the cytoplasm or as a membrane associated protein [114], and is sometimes detected in lysosome targeted endosomes colocalizing with Rab7 and partially with lamp-1 [123]. On the contrary, in HeLa cells stimulated with sorbitol, P62 has been detected in the nucleus consistent with its suggested role as a transcription factor [137]. For example, P62 has been shown to potentiate transactivation by chicken
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CHAPTER 1. INTRODUCTION
Figure 1.3:Schematic domain organization of murine P62
ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily of ligand-activated transcription factors [89]. As P62 lacks any classical nuclear localization signals itself, it is supposed to shuttle into the nucleus bound to adaptor proteins [40, 109]. In earlier publications, P62 has been assumed to possess either serine/threonine kinase activity [107], or, due to a number of putative target sites for several protein kinases, to be a substrate for phosphorylation itself [114]. Today, the general notion prevails, that P62 is neither a kinase nor the substrate of a kinase, but acts as a scaffold protein or a molecular adaptor, linking the functionality of otherwise promiscous cellular enzymes to discrete signal transduction events. The binding of P62 to atypical protein kinases (aPKCs), namely PKCζand PKCλ/ι, seems to be of outstanding functional relevance. The aPKCs share a conserved catalytic domain with the classical and novel PKCs, but possess a clearly distinct regulatory do-main [65, 98]. They are insensitive to Ca2+as well as diacylglycerol and phorbol esters, and in contrast to other family members, they contain only one zinc finger domain and no C2 domain. On a functional level, aPKCs are essential components of signal transduction cascades regulating cell growth and survival [7, 29], as well as cytoskeletal rearrange-ments [149] and cell polarity [83]. The interaction of P62 with aPKCs, especially PKCζ, has been reported by independent groups [17, 114, 123], and the interacting domains have been mapped using deletional mutants: P62 utilizes a novel acidic motif comprising amino acids 69 to 81 (AID domain) for the interaction [124], whereas the regulatory domain of PKCζ(PB1 domain) is involved. More recently, the solution structure of aPKC’s PB1 do-main and its mode of interaction with P62 was determined [57]. Furthermore, the crystal structure of the interacting motifs was solved [157], confirming earlier results regarding this interaction. The interaction of P62 and PKCζhas been shown to target PKC activity to several path-ways and cellular functions. For example, P62 links the Kvβ2 subunit of the voltage-gated potassium channel as well as theρsubunit of the ligand-gated ion channel GABACR to PKCζ[24, 46]. In 2002 Burnolet al.showed that growth factor receptor-bound pro-tein 14 (Grb14), a negative regulator of insulin signaling, is phosphorylated by PKCζ as a consequence of the interaction of Grb14 and the heterodimeric P62/PKCζcomplex. As phosphorylated Grb14 is an even stronger inhibitor of insulin signaling, the cellular metabolism is influenced by the scaffold protein P62 [17].
1.3. LINK BETWEEN IL-4R AND P62
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Selective activation of the transcription factor nuclear factorκB (NF-κB) in response to different extracellular stimuli is another funtion of P62, which has been extensively studied. For example, signals induced by nerve growth factor (NGF) are transduced via the neurotrophin receptors p75 and TrkA and result in P62 mediated NF-κB activation. P62 directly binds to TrkA, and to p75 using TRAF6 as a molecular bridge. PKCζ, that is recruited to this macromolecular complex, phosphorylates IκB kinase, leading to phosphorylation and degradation of inhibitor ofκB (IκB) and subsequent activation of NF-κ P62B, which results in cell survival.also been shown to participate in TrkA has internalization, connecting receptor signals with the endosomal signaling network required for neuronal differentiation [41, 67, 122, 159]. In a similar manner, signal transduction processes in response to the cytokines tumor necrosis factor-α(TNF-α binds to RIP, a death P62) and IL-1 are influenced by P62: domain kinase that associates with the TNF receptor 1 (TNF-R1) through interaction with the adaptor molecule TRAAD, thereby recruiting PKCζ a result, IKK. Asβ-kinase functionality (mediated by PKCζ) is targeted to the TNF receptor [81, 125]. the Likewise, IL-1 receptor is connected via myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor associated kinase (IRAK) and TRAF6 to the scaffold protein P62, which recruits PKCζand consequently causes NF-κB activation upon stimulation with IL-1 [124]. The involvement of P62 in these signal transduction pathways has been investigated mainly in cell culture systems employing siRNA approaches or using dominant negative variants of participating proteins. To verify these oberservations, a P62 gene-deficient mouse model was established. The first study on this mouse model reported an im-paired induced osteoclastogenesis in P62 gene-deficient mice, whereas the basal physiol-ogy of bones was not affected [33]. Stimulation with the calciotropic hormone parathyroid hormone-related protein (PTHrP) induces osteoclastogenesis via the receptor activator of NF-κB ligand (RANK-L), which is a member of the TNF-α Engage-family of cytokines. ment of RANK leads to the formation of a ternary complex of TRAF6, P62 and aPKC. Most likely, PKCλ/ιis involved in this complex formation, since PKCζgene-deficient mice displayed no osteopetrotic phenotype. Recently, Rodriguezet al.reported that the loss of P62 leads to mature-onset obesity [116]. Mice older than five months displayed increased body fat, a reduced metabolic rate and insulin- as well as leptin-resistance. Most likely this phenotype is due to an enhanced basal activity of the extracellular signal-regulated protein kinase (ERK) in adipose tissue, which has been shown to be important for adipo-genesis and obesity [12]. P62 can directly interact with ERK, and in a WT situation this interaction seems to block ERK activity, resulting in reduced adipocyte differentiation.
1.3 Link between IL-4R and P62 Both IRS- and STAT6-independent IL-4 induced proliferation in a TF1 cell culture system was observed in the laboratory of Prof. Gessner. As a logical consequence, we postulated that a third signal transduction pathway for IL-4R signals, independent of the signal transduction proteins described until now, must exist. To identify proteins involved in this hypothetical signal pathway, a yeast two hybrid (Y2H) screen was performed. In
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