USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals from endosomes.

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.

Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection.

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.

A possible function of Nik-related kinase in the labyrinth layer of delayed delivery mouse placentas.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2-PTEN-AKT Pathway and Placental Cell Proliferation.

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain.

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing's disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645-684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing's disease due to USP8 mutations.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

Nik-related kinase is targeted for proteasomal degradation by the chaperone-dependent ubiquitin ligase CHIP.

Nik-related kinase (Nrk) is a member of the germinal center kinase IV family and suppresses Akt signaling. In vivo, Nrk prevents placental hyperplasia and breast cancer formation. Here, we show that Nrk is regulated by the chaperone-dependent ubiquitin ligase carboxyl terminus of heat-shock protein (Hsp)70-interacting protein (CHIP). Immunoprecipitation and liquid chromatography-tandem mass spectrometry analysis reveal that Nrk preferentially interacts with CHIP and Hsp70/90 family proteins. Nrk protein levels are decreased by CHIP overexpression and increased by siRNA-mediated CHIP knockdown. Our results indicate that Nrk is ubiquitinated by CHIP in a chaperone-dependent manner, resulting in its proteasomal degradation. CHIP targets a fraction of Nrk molecules that have lost the ability to regulate Akt signaling. We conclude that CHIP plays an important role in regulating Nrk protein levels.

RNA-seq of Isolated Chromaffin Cells Highlights the Role of Sex-Linked and Imprinted Genes in Adrenal Medulla Development.

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors.

Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.

IRS-2 deubiquitination by USP9X maintains anchorage-independent cell growth via Erk1/2 activation in prostate carcinoma cell line.

Insulin-like growth factors (IGFs) have been shown to induce proliferation of many types of cells. Insulin receptor substrates (IRSs) are major targets of IGF-I receptor (IGF-IR) tyrosine kinase activated by IGFs, and are known to play important roles in the activation of downstream signaling pathways, such as the Erk1/2 pathway. Dysregulation of IGF signaling represents a central tumor promoting principle in human carcinogenesis. Prostate carcinoma is highly dependent on the IGF/IGF-IR/IRS axis. Here we identified the deubiquitinase, ubiquitin specific peptidase 9X (USP9X) as a novel binding partner of IRS-2. In a human prostate carcinoma cell line, small interfering RNA (siRNA)-mediated knockdown of USP9X reduced IGF-IR as well as IRS-2 protein levels and increased their ubiquitination. Knockdown of USP9X suppressed basal activation of the Erk1/2 pathway, which was significantly restored by exogenous expression of IRS-2 but not by IGF-IR, suggesting that the stabilization of IRS-2 by USP9X is critical for basal Erk1/2 activation. Finally, we measured anchorage-independent cell growth, a characteristic cancer feature, by soft-agar colony formation assay. Knockdown of USP9X significantly reduced anchorage-independent cell growth of prostate carcinoma cell line. Taken all together, our findings indicate that USP9X is required for the promotion of prostate cancer growth by maintaining the activation of the Erk1/2 pathway through IRS-2 stabilization.

Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion.

Deubiquitylases USP5 and USP13 are recruited to and regulate heat-induced stress granules through their deubiquitylating activities.

Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper.

Reduced SHARPIN and LUBAC Formation May Contribute to CCl4- or Acetaminophen-Induced Liver Cirrhosis in Mice.

Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl4) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.

USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

Insulin receptor substrates (IRSs) are phosphorylated by IGF-I receptor tyrosine kinase in a ligand-dependent manner. In turn, they bind to and activate effector proteins such as PI3K, leading to various cell responses including cell proliferation. We had reported that ubiquitin ligase Nedd4 induces mono-ubiquitination of IRS-2, thereby enhancing IRS-2 tyrosine phosphorylation, leading to increased IGF signaling and mitogenic activity. Here we show that ubiquitin-specific protease 15 (USP15) antagonizes the effect of Nedd4 on IRS-2. We identified USP15 as a protein that preferentially bound to IRS-2 when IRS-2 was conjugated with ubiquitin. In HEK293 cells, Nedd4 overexpression induced IRS-2 ubiquitination, which was decreased by USP15 co-expression while increased by USP15 knockdown. Nedd4 overexpression enhanced IGF-I-dependent IRS-2 tyrosine phosphorylation, and USP15 co-expression suppressed it. Conversely, USP15 knockdown increased IRS-2 tyrosine phosphorylation and downstream signaling in prostate cancer PC-3 cells. We concluded that USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

Glucose deprivation attenuates sortilin levels in skeletal muscle cells.

In skeletal muscle, sortilin plays a predominant role in the sorting of glucose transporter 4 (Glut4), thereby controlling glucose uptake. Moreover, our previous study suggested that the sortilin expression levels are also implicated in myogenesis. Despite the importance of sortilin in skeletal muscle, however, the regulation of sortilin expression has not been completely understood. In the present study, we analyzed if the sortilin expression is regulated by glucose in C2C12 myocytes and rat skeletal muscles in vivo. Sortilin protein expression was elevated upon C2C12 cell differentiation and was further enhanced in the presence of a high concentration of glucose. The gene expression and protein degradation of sortilin were not affected by glucose. On the other hand, rapamycin partially reduced sortilin induction by a high concentration of glucose, which suggested that sortilin translation could be regulated by glucose, at least in part. We also examined if the sortilin regulation by glucose was also observed in skeletal muscles that were obtained from fed or fasted rats. Sortilin expression in both gastrocnemius and extensor digitorum longus (EDL) muscle was significantly decreased by 17-18h of starvation. On the other hand, pathological levels of high blood glucose did not alter the sortilin expression in rat skeletal muscle. Overall, the present study suggests that sortilin protein levels are reduced under hypoglycemic conditions by post-transcriptional control in skeletal muscles.

Deficiency of X-Linked Protein Kinase Nrk during Pregnancy Triggers Breast Tumor in Mice.

The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.

Involvement of resistin-like molecule ß in the development of methionine-choline deficient diet-induced non-alcoholic steatohepatitis in mice.

Resistin-like molecule ß (RELMß) reportedly has multiple functions including local immune responses in the gut. In this study, we investigated the possible contribution of RELMß to non-alcoholic steatohepatitis (NASH) development. First, RELMß knock-out (KO) mice were shown to be resistant to methionine-choline deficient (MCD) diet-induced NASH development. Since it was newly revealed that Kupffer cells in the liver express RELMß and that RELMß expression levels in the colon and the numbers of RELMß-positive Kupffer cells were both increased in this model, we carried out further experiments using radiation chimeras between wild-type and RELMß-KO mice to distinguish between the contributions of RELMß in these two organs. These experiments revealed the requirement of RELMß in both organs for full manifestation of NASH, while deletion of each one alone attenuated the development of NASH with reduced serum lipopolysaccharide (LPS) levels. The higher proportion of lactic acid bacteria in the gut microbiota of RELMß-KO than in that of wild-type mice may be one of the mechanisms underlying the lower serum LPS level the former. These data suggest the contribution of increases in RELMß in the gut and Kupffer cells to NASH development, raising the possibility of RELMß being a novel therapeutic target for NASH.

Prolyl isomerase Pin1 negatively regulates AMP-activated protein kinase (AMPK) by associating with the CBS domain in the γ subunit.

AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the ß subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr(211)-Pro-containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, whereas Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1, and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPKα subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes, such as PGC-1α, which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation in the muscles of Pin1 KO mice as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK.

LUBAC Formation Is Impaired in the Livers of Mice with MCD-Dependent Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic lipid accumulation followed by the inflammation-induced death of hepatocytes and fibrosis. In this process, oxidative stress contributes to the induction of several inflammatory cytokines including TNF-α andIL-1ß in macrophages, while, in hepatocytes, NF-κB reportedly induces the expressions of cell survival genes for protection from apoptosis. Recently, it was reported that the new ubiquitin ligase complex termed linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on NF-κB essential modulator (NEMO) and thereby induces NF-κB pathway activation. In this study, we demonstrated the formation of LUBAC to be impaired in the livers of NASH rodent models produced by methionine and choline deficient (MCD) diet feeding, first by either gel filtration or Blue Native-PAGE, with subsequent confirmation by western blotting. The reduction of LUBAC is likely to be attributable to markedly reduced expression of SHARPIN, one of its components. Thus, impaired LUBAC formation, which would result in insufficient NF-κB activation, may be one of the molecular mechanisms underlying the enhanced apoptotic response of hepatocytes in MCD diet-induced NASH livers.

The Novel Functions of High-Molecular-Mass Complexes Containing Insulin Receptor Substrates in Mediation and Modulation of Insulin-Like Activities: Emerging Concept of Diverse Functions by IRS-Associated Proteins.

Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases.