Mechanism of p38 MAPK–induced EGFR endocytosis and its crosstalk with ligand-induced pathways

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.     

Analysis of the acrolein-modified sites of apolipoprotein B-100 in LDL

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 μM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH₂-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.     

Identification of a Metabolizing Enzyme in Human Kidney by Proteomic Correlation Profiling

Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named “proteomic correlation profiling” in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein''s correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology.Molecular identification for an enzyme reaction or biologically active substance in an organism is challenging, although molecular biological methodologies such as expression cloning (1), recombinant protein panel (2) and RNAi screening (3) have been introduced recently as alternative approaches. Conventional biochemical purification has provided a number of successes and thus still remains a powerful, though labor-intensive strategy.In the traditional protein purification, it had been necessary to purify an individual protein nearly to homogeneity at a microgram amount so that the purified protein could be analyzed by N-terminal amino acid sequencing. Protein identification by mass spectrometry subsequently revolutionized this technology by enabling identification of proteins at much lower abundances: individual proteins could then be associated with specific activities as soon as a band in SDS-PAGE could be observed, even when the purified protein was far from homogeneity (4–6). Although this streamlined the workflow by reducing the required starting materials as well as the separation steps for protein purification, a faster and more generalized approach from smaller starting material has still been desired because some proteins are physiochemically difficult for example in solubilization and stability. To solve these problems, we devised a proteomic correlation profiling methodology (7).The basic concept of proteomic correlation profiling was originally developed by Andersen et al. (8). They quantitatively profiled hundreds of proteins across several centrifugation fractions by mass spectrometry and identified centrosomal proteins by calculating the correlation of these protein expression profiles with already known centrosomal proteins. In the following study, Foster et al. applied this strategy to map more than 1400 proteins to ten subcellular locations (9). Although these studies used centrifugation as a separation method and a known marker profile as a standard for correlation, we extended this concept to use chromatography as a separation method and kinase activity as a basis for comparison; our approach successfully identified a kinase responsible for phosphorylation of peptide substrates just after one step chromatography, and was termed proteomic correlation profiling (7). Independently, Kuromitsu et al. reported identification of an active substance in the serum response element-dependent luciferase assay from interstitial cystitis urine after three-step chromatography by a similar concept (10). In theory, this general proteomic correlation profiling strategy can be adapted to any kind of separation method and activity profile but no other example has been reported thus far, therefore, actual examples where the method can be applied to other enzyme classes are required to prove its generality.Multiple sclerosis is the most common autoimmune disorder of the central nerve system in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring (11, 12). Until recently, the standard treatments for multiple sclerosis such as interferon beta, glatiramer acetate, mitoxantrone, and natalizumab would often cause severe adverse events (13, 14), providing an opportunity for development of less dangerous treatments for this disease. However, in 2010, Food and Drug Administration approved fingolimod (Gilenya; chemical structure in Fig. 1) as the first oral medicine, and recommended this as a first-line treatment for relapsing-remitting multiple sclerosis, opening up a new therapeutic approach to the disease (15).Open in a separate windowFig. 1.The chemical structures of CS-0777, fingolimod and their phosphorylated derivatives.Sphingosine 1-phosphate receptor 1 (S1P1)¹ modulators are emerging as a new class of drugs with potential therapeutic application in multiple sclerosis (15), and fingolimod is a nonselective sphingosine 1-phosphate (S1P) receptor modulator (16–18, 21, 22). Given its structural similarity to sphingosine, fingolimod is phosphorylated in vivo by sphingosine kinase, in particular sphingosine kinase 2 (SPHK2) (19, 20), and the fingolimod-phosphate (fingolimod-P, Fig. 1) binds to and activates four G protein-coupled S1P receptors (21, 22). By this mechanism, fingolimod-P induces internalization of S1P1 on lymphocytes, blocking the ability of the receptor to support lymphocyte egress and recirculation through secondary lymphoid organs. This suppresses immune responses and is presumably the main immunomodulatory mode of action of fingolimod.CS-0777 (Fig. 1) is a novel selective S1P1 modulator (23). Although the immunomodulatory effects are supposed to be mainly mediated by S1P1, some lines of evidence suggest that the agonist activity on S1P receptor 3 (S1P3) could cause acute toxicity and cardiovascular deregulation, including bradycardia in rodents (24, 25). Thus, CS-0777 was designed to have more selectivity on S1P1 over S1P3 in contrast to fingolimod-P which has potent agonistic activity for S1P3, S1P4, and S1P5 in vitro (22). Like fingolimod, CS-0777 is also a prodrug phosphorylated in vivo, and the phosphorylated CS-0777 (CS-0777-P, Fig. 1) agonizes S1P1 with more than 300-fold selectivity relative to S1P3 whereas CS-0777-P has weaker effects on S1P5 and no activity on S1P2 (23). CS-0777 showed immunosuppressive activity in mouse and rat models of experimental autoimmune encephalitis, animal models for multiple sclerosis. In healthy volunteers, single oral doses of CS-0777 caused marked, dose-dependent decreases in numbers of circulating lymphocytes, including marked and reversible decreases in circulating T and B cells (26). Furthermore, in multiple sclerosis patients, single oral doses of CS-0777 caused dose-dependent decreases in circulating lymphocytes, with a slightly greater suppression of CD4+ versus CD8+ T cells. Therefore, CS-0777 would alter immune responses solely through activation of S1P1 without S1P3 modulation in humans, which could circumvent a bradycardia adverse effect, although the relationships associating selectivity of S1P1 to S1P3 with bradycardia in humans are not fully understood (12).Orally administrated CS-0777 is phosphorylated and rapidly reaches equilibrium with CS-0777-P as in the case of fingolimod (22), suggesting that the high kinase activity in blood is balanced by phosphatases. Therefore, identification of a phosphatase, the inactivating enzyme of an active metabolite, as well as identification of a kinase, the activating enzyme of a prodrug, are critical to fully understand the mechanism of action at the molecular level for both CS-0777 and fingolimod. Sphingosine kinase 2 (SPHK2) was identified as the major kinase of fingolimod (21, 28, 29) and lipid phosphate phosphatase 3 (LPP3) was reported to be a phosphatase for fingolimod-P dephosphorylation (30), although contribution of LPP3 in vivo has not been fully studied. In our previous work, we have identified CS-0777 kinases in human blood as fructosamine 3-kinase-related protein (FN3K-RP) and fructosamine 3-kinase (FN3K) (6), whereas the phosphatase of CS-0777-P had not been identified thus far.In this study, we have successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as the major CS-0777-P phosphatase candidate in the human kidney by proteomic correlation profiling. According to available information, this is the first report applying proteomic correlation profiling to enzyme classes other than kinases; similarly, we believe this to be first application of proteomic correlation profiling to human tissue extract, which therefore has opened up wide usage of proteomic correlation profiling for all types of enzyme identification.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca²⁺-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-β-d-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 ⁺ with concomitant resumption of the mutant cells’ growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.     

Receptor-like cytoplasmic kinases are pivotal components in pattern recognition receptor-mediated signaling in plant immunity

Innate immunity is generally initiated with recognition of conserved pathogen-associated molecular patterns (PAMPs). PAMPs are perceived by pattern recognition receptors (PRRs), leading to activation of a series of immune responses, including the expression of defense genes, ROS production and activation of MAP kinase. Recent progress has indicated that receptor-like cytoplasmic kinases (RLCKs) are directly activated by ligand- activated PRRs and initiate pattern -triggered immunity (PTI) in both Arabidopsis and rice. To suppress PTI, pathogens inhibit the RLCKs by many types of effectors, including AvrAC, AvrPphB and Xoo1488. In this review, we summarize recent advances in RLCK-mediated PTI in plants.     

Mutation of GDP-Mannose-4,6-Dehydratase in Colorectal Cancer Metastasis

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.