Abstract Functional analysis of the immunoreceptor tyrosine‐based activation motif (ITAM) derived from the membrane‐proximal ITAM of CD3ζ demonstrates that mutations at either the tyrosine or leucine residues in the N‐terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C‐terminal YxxL segment abrogate signals for interleukin (IL)‐2 production but do not prevent tyrosine phosphorylation of the N‐terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C‐γl or calcium mobilization. Cross‐linking of chimeric receptors containing a C‐terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL‐2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL‐2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N‐terminal YxxL segment reduces the receptor cross‐linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross‐linking required to trigger tyrosine phosphorylation‐dependent signaling events.
An increase in intracellular cAMP level induced the expression of IL-2R alpha-chain, the 55-kDa component of IL-2R complex, in a human NK-like cell line, YT. We show here that forskolin also induces the expression of IL-2R alpha-chain on mouse large granular lymphocytes (LGL) but not on T cells. In contrast, treatment with a combination of phorbol ester and calcium ionophore, which is a strong inducer of IL-2R alpha-chain on T cells, does not induce the expression of the alpha-chain on LGL cells. Forskolin was shown to activate the transcription of IL-2R alpha-chain gene in YT cells as revealed by the chloramphenicol acetyltransferase assay. Chemical cross-linking experiments using radio-iodinated IL-2 also supported the enhanced expression of IL-2R alpha-chain by treatment with forskolin. In contrast to the alpha-chain, IL-2R beta-chain was not induced by forskolin as revealed by flow cytofluorometry with a mAb against the beta-chain molecule. These results indicate that the activation of adenylate cyclase induces or/and enhance the expression of IL-2R alpha-chain at the transcriptional level in LGL/NK cells including mouse LGL and human YT cell, which leads to the enhanced expression of high affinity IL-2 receptors.
Natural helper (NH) cells, a member of Lin(-)IL-2R(+)IL-7R(+)IL-25R(+)IL-33R(+)GATA3(+) group 2 innate lymphoid cell subset, are characterized by the expression of transcription factors GATA3 and RORα and production of large amounts of Th2 cytokines such as IL-5, IL-6, and IL-13 upon IL-33 stimulation or a combination of IL-2 and IL-25. We have studied the signal transduction pathways critical for the cytokine expression and development of NH cell. Either stimulation with IL-33 or a combination of IL-2 and IL-25 induced p38 activation and phosphorylation of GATA3 in NH cells, and the phosphorylated form of GATA3 bound to the IL-5 and IL-13 promoters. All these events were blocked by SB203580, a p38 inhibitor. Inhibition of p38 also blocked IL-6 production. The mature NH cells lacking Gata3 were impaired in the proliferation and production of IL-5 and IL-13, but not IL-6, indicating that both p38 and GATA3 are critical for the proliferation and production of IL-5 and IL-13 and that the mechanisms downstream of p38 differ between IL-6 and IL-5/IL-13. In contrast, the NH cells lacking RORα showed no impairment in the proliferation and cytokine production, indicating that GATA3 but not RORα plays a pivotal role in the effector functions of mature NH cell. However, deletion of either GATA3 or RORα in hematopoietic stem cells severely blocked the development into NH cells. Our results demonstrate the important roles of p38 and GATA3 in NH cell functions.
Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6-related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and beta-galactosidase (Adbeta-gal). Compared with cardiomyocytes infected with Adbeta-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Adbeta-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal alpha-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal-regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Adbeta-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.
Antigen cross-presentation is critical in infectious and tumor immunity where cytotoxic T lymphocytes are induced by dendritic cells specifically equipped with cellular machineries to present exogenous antigens with major histocompatibility complex (MHC) class I molecules. To examine molecular mechanisms of antigen cross-presentation, we employed as a model system a murine dendritic cell line DC2.4 capable of presenting soluble antigens such as ovalbumin (OVA) with MHC class I. Here, we demonstrate that exogenously added OVA is accumulated in the endoplasmic reticulum (ER) and late endosomes followed by retrograde transport to the cytoplasm through the Sec61 transporter complexes, and that CHIP functions as an E3 ubiquitin–ligase for OVA degradation by proteasomes. This mechanism is essentially the same as that known as the ER-associated degradation (ERAD) in the quality control of secretary and membrane proteins.
Endoplasmic-reticulum-associated protein degradation
Deoxyribonucleic acid (DNA) phage phi CbK-resistant nonmotile mutants of Caulobacter crescentus CB15 were examined for their formation of polar surface structures (a stalk, a single flagellum, pili, and DNA phage receptors). These mutants were devoid of pili and DNA phage receptors and simultaneously defective either in both stalk formation and flagellar activity (stalk-defective type) or in the formation of normal flagella (flagella-defective type). DNA phage phi Cr30-mediated transductions revealed that stalk-defective mutants were of a single genetic type, whereas flagella-defective mutants were grouped into two different genetic types, I and II. To investigate how membrane proteins change in the above morphology mutants, cell envelopes pulse-labeled with L-[35S]methionine were analyzed by two-dimensional gel electrophoresis. No gross change of membrane proteins was observed in the stalk-defective mutant CB15 pdr-803, except a 49,000-molecular-weight (49K) protein which was found reduced. However, a 27K, two 28.5K, and a 70.5K protein were missing from the membrane of the flagella-defective type I mutant CB15 pdr-813. These proteins are most likely to be flagella-related protein, flagellins A and B, and hook protein, respectively. In another flagella-defective type II mutant, CB15 pdr-816, the 27K and two 28.5K proteins were similarly absent but the 70.5K protein was consistently present in the membrane. The synthesis of flagellin was next assayed radioimmunologically in the above 35S-labeled mutants. Stalk-defective CB15 pdr-803 synthesized flagellin normally, compared to the wild-type strain. Flagellins A (26K) and B (28K) formed multiple spots in isoelectric focusing. A 29K protein was also detected in the flagellin-specific radioactivity from the cytoplasm. Flagella-defective type I CB15 pdr-813 synthesized flagellin only at a basal level. Thus transcription or translation of flagellin appeared to be repressed in this mutant. Another flagella-defective type II strain, CB15 pdr-816, however, synthesized flagellin at an apparently enhanced rate compared with the wild type. Flagellin synthesized in CB15 pdr-816 was flagellin A and a smaller 22K flagellin. Flagellin B was not synthesized in the mutant. It then follows that flagellin B is not a precursor of flagellin A and the 22K flagellin. Flagella-defective type II CB15 pdr-816, without flagellin B, formed a stub structure with a hook attached to one end instead of normal flagella. In the wild-type membrane, flagellin B was the major flagellin, whereas flagellin A was major in the cytoplasm and the flagellar filament. It is suggested from these results that flagellin B is important in the assembly of normal flagella.
