Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence.

Heterochromatin, a key component of the eukaryotic nucleus, is fundamental to the regulation of genome stability, gene expression and cellular functions. However, the factors and mechanisms involved in heterochromatin formation and maintenance still remain largely unknown. Here, we show that insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein, is indispensable for constitutive heterochromatin formation via liquid‒liquid phase separation (LLPS). In particular, IRTKS droplets can infiltrate heterochromatin condensates composed of HP1α and diverse DNA-bound nucleosomes. IRTKS can stabilize HP1α by recruiting the E2 ligase Ubc9 to SUMOylate HP1α, which enables it to form larger phase-separated droplets than unmodified HP1α. Furthermore, IRTKS deficiency leads to loss of heterochromatin, resulting in genome-wide changes in chromatin accessibility and aberrant transcription of repetitive DNA elements. This leads to activation of cGAS-STING pathway and type-I interferon (IFN-I) signaling, as well as to the induction of cellular senescence and senescence-associated secretory phenotype (SASP) responses. Collectively, our findings establish a mechanism by which IRTKS condensates consolidate constitutive heterochromatin, revealing an unexpected role of IRTKS as an epigenetic mediator of cellular senescence.

Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4.

Missing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAi-mediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins.

IRTKS Promotes Insulin Signaling Transduction through Inhibiting SHIP2 Phosphatase Activity.

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT-mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT-mTOR signaling pathway to modulate cell proliferation.

IRTKS is correlated with progression and survival time of patients with gastric cancer.

BACKGROUND AND OBJECTIVES: IRTKS functions as a novel regulator of tumour suppressor p53; however, the role of IRTKS in pathogenesis of gastric cancer is unclear. DESIGN: We used immunohistochemistry to detect IRTKS levels in 527 human gastric cancer specimens. We generated both IRTKS-deficient and p53-deficient mice to observe survival time of these mice and to isolate mouse embryonic fibroblasts (MEFs) for evaluating in vivo tumorigenicity. Co-immunoprecipitation was used to study the interaction among p53, MDM2 and IRTKS, as well as the ubiquitination of p53. RESULTS: IRTKS was significantly overexpressed in human gastric cancer, which was conversely associated with wild-type p53 expression. Among patients with wild-type p53 (n=206), those with high IRTKS expression (n=141) had a shorter survival time than those with low IRTKS (n=65) (p=0.0153). Heterozygous p53+/- mice with IRTKS deficiency exhibited significantly delayed tumorigenesis and an extended tumour-free survival time. p53+/- MEFs without IRTKS exhibited attenuated in vivo tumorigenicity. IRTKS depletion upregulated p53 and its target genes, such as BAX and p21. Intriguingly, IRTKS overexpression promoted p53 ubiquitination and degradation in MEFs and gastric cancer cells. Under DNA damage conditions, IRTKS was phosphorylated at Ser331 by the activated Chk2 kinase and then dissociated from p53, along with the p53-specific E3 ubiquitin ligase MDM2, resulting in attenuated p53 ubiquitination and degradation. CONCLUSION: IRTKS overexpression is negatively correlated with progression and overall survival time of patients with gastric cancer with wild-type p53 through promotion of p53 degradation via the ubiquitin/proteasome pathway.

The Rho GTPase Rif signals through IRTKS, Eps8 and WAVE2 to generate dorsal membrane ruffles and filopodia.

Rif induces dorsal filopodia but the signaling pathway responsible for this has not been identified. We show here that Rif interacts with the I-BAR family protein IRTKS (also known as BAIAP2L1) through its I-BAR domain. Rif also interacts with Pinkbar (also known as BAIAP2L2) in N1E-115 mouse neuroblastoma cells. IRTKS and Rif induce dorsal membrane ruffles and filopodia. Dominant-negative Rif inhibits the formation of IRTKS-induced morphological structures, and Rif activity is blocked in IRTKS-knockout (KO) cells. To further define the Rif-IRTKS signaling pathway, we identify Eps8 and WAVE2 (also known as WASF2) as IRTKS interactors. We find that Eps8 regulates the size and number of dorsal filopodia and membrane ruffles downstream of Rif-IRTKS signaling, whereas WAVE2 modulates dorsal membrane ruffling. Furthermore, our data suggests that Tir, a protein essential for enterohemorrhagic Escherichia coli infection, might compete for Rif for interaction with the I-BAR domain of IRTKS. Based on this evidence, we propose a model in which Rho family GTPases use the I-BAR proteins, IRSp53 (also known as BAIAP2), IRTKS and Pinkbar, as a central mechanism to modulate cell morphology.

The SH3 domain distinguishes the role of I-BAR proteins IRTKS and MIM in chemotactic response to serum.

The family of inverse BAR (I-BAR) domain proteins participates in a range of cellular processes associated with membrane dynamics and consists of five distinct members. Three of the I-BAR proteins, including insulin receptor tyrosine kinase substrate (IRTKS), contain an SH3 domain near their C-termini. Yet, the function of the SH3 domain of IRTKS remains uncharacterized. Here we report that in contrast to MIM, which is a prototype of I-BAR proteins and does not contain an SH3 domain, IRTKS promoted serum-induced cell migration along with enhanced phosphorylation of mitogen activated kinases Erk1/2 and p38, and activation of small GTPases Rac1 and Cdc42. In addition, cells overexpressing IRTKS exhibited an increased polarity characterized by elongated cytoplasm and extensive lamellipodia at leading edges. However, a mutant with deletion of the SH3 domain attenuated both cellular motility and p38 phosphorylation but had little effect on Erk1/2 phosphorylation. Also, a chimeric mutant in which the N-terminal portion of MIM is fused with the C-terminal IRTKS, including the SH3 domain, was able to promote chemotactic response to serum and cellular polarity. In contrast, a chimeric mutant in which the N-terminal IRTKS is fused with the C-terminal MIM failed to do so. Furthermore, treatment of cells with SB203580, a selective inhibitor of p38, also neutralized the effect of IRTKS on cell migration. These data indicate that the SH3 domain distinguishes the function of IRTKS in promoting cell migration and inducing signal transduction from those of SH3-less I-BAR proteins.

IRTKS promotes osteogenic differentiation by inhibiting PTEN phosphorylation.

Insulin stimulates osteoblast proliferation and differentiation as an anabolic agent in bone. Insulin Receptor Tyrosine Kinase Substrate (IRTKS) is involved in insulin signaling as an adapter for insulin receptors (IR). Here, we showed that IRTKS levels were significantly decreased in bone marrow mesenchymal stem cells (BMSCs) derived from the bone marrow of patients with osteoporosis. Based on relevant experiments, we observed that IRTKS promoted the proliferation, migration, and osteoblast differentiation of BMSCs and MC3T3-E1 cells. In addition, we identified a Phosphatase and Tensin homolog deleted on chromosome 10 (PTEN) as a potential active substrate of IRTKS. We demonstrated a direct interaction between IRTKS and PTEN using co-immunoprecipitation. Subsequently, we confirmed that the SH3 domain of IRTKS directly binds to the C-terminal tail of PTEN. Further experimental results demonstrated that PTEN attenuated the promoting effects of IRTKS on the proliferation, migration, and osteoblast differentiation of BMSCs and MC3T3-E1 cells. In conclusion, this study suggests that IRTKS contributes to osteogenic differentiation by inhibiting PTEN phosphorylation and provides a potential therapeutic target for osteoporosis patients.

IRTKS contributes to the malignant progression of cervical cancer cells.

Cervical cancer (CC), one of the most aggressive tumors in women, has high risk rates of recurrence and metastasis. It is essential to study the key genes and proteins involved in CC development. IRTKS, a member of the IRSp53 family, has been reported as a tumor promoter in gastric and breast cancers. However, the biological role of IRTKS in CC is still unclear. The purpose of this study was to explore the biological function of IRTKS in CC cells in vitro and the effect of IRTKS on tumorigenesis in vivo. Siha and Hela cells were treated with si-RNA and plasmids. Cell proliferation and growth were detected by CCK8, colony formation assay and nude mouse tumorigenicity assay, respectively. Transwell assay was used to analyze cell migration and invasion. The expression of epithelial-mesenchymal transition (EMT)-related proteins was determined by western blot. IRTKS was highly expressed in CC. IRTKS contributed to the proliferation of CC cells in vitro and in vivo. Furthermore, IRTKS facilitated the migration and invasion of CC cells and modulated EMT. IRTKS plays a crucial role in CC tumorigenesis, suggesting it may be a potential key gene for new therapeutic strategies in CC.

Cooperation between IRTKS and deubiquitinase OTUD4 enhances the SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression.

Elevated expression and genetic aberration of IRTKS, also named as BAIAP2L1, have been observed in many tumors, especially in tumor progression. however, the molecular and cellular mechanisms involved in the IRTKS-enhanced tumor progression are obscure. Here we show that higher IRTKS level specifically increases histone H3 lysine 9 trimethylation (H3K9me3) by promoting accumulation of the histone methyltransferase SETDB1. Furthermore, we reveal that IRTKS recruits the deubiquitinase OTUD4 to remove Lys48-linked polyubiquitination at K182/K1050 sites of SETDB1, thus blocking SETDB1 degradation via the ubiquitin-proteasome pathway. Interestingly, the enhanced IRTKS-OTUD4-SETDB1-H3K9me3 axis leads to a general decrease in chromatin accessibility, which inhibits transcription of CDH1 encoding E-cadherin, a key molecule essential for maintaining epithelial cell phenotype, and therefore results in epithelial-mesenchymal transition (EMT) and malignant cell metastasis. Clinically, the elevated IRTKS levels in tumor specimens correlate with SETDB1 levels, but negatively associate with survival time. Our data reveal a novel mechanism for the IRTKS-enhanced tumor progression, where IRTKS cooperates with OTUD4 to enhance SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression. This study also provides a potential approach to reduce the activity and stability of the known therapeutic target SETDB1 possibly through regulating IRTKS or deubiquitinase OTUD4.

Insulin receptor tyrosine kinase substrate (IRTKS) promotes the tumorigenesis of pancreatic cancer via PI3K/AKT signaling.

Pancreatic cancer (PC) is a common type of tumor, which ranks for the seventh leading cause of cancer death worldwide. Insulin receptor tyrosine kinase substrate (IRTKS) plays an important regulatory role in cell proliferation, motility and survival. In this study, we explore the effect of IRTKS on the occurrence and development of PC. The expression and clinical features of IRTKS were predicted in database, PC cell lines and samples. IRTKS overexpressed and knocked down PC cell lines were established by lentivirus. CCK-8 assay, scratch migration assay and Transwell assay were used to analyze IRTKS oncogenic functions in cell lines. Bioinformatic enrichment analysis were conducted to explore the biological functions IRTKS involved in PC and Western Bolt assay was performed to reveal the downstream signaling molecules. It is detected that IRTKS is highly expressed in PC (P < 0.05), and overexpression of IRTKS predicted worse overall survival (OS, P = 0.018). The proliferation, migration and invasion ability were significantly enhanced in IRTKS overexpressed cells and inhibited in IRTKS knocked down cells (P < 0.05). Bioinformatic enrichment analysis based on GSE46583 dataset showed that IRTKS was significantly involved in PI3K/AKT pathway. Further investigation revealed that overexpression of IRTKS upregulated the ratio of p-PI3K/PI3K and p-AKT/AKT in vitro, while silencing of IRTKS presented opposite results, and PI3K inhibitor LY294002 treatment induced the phenotypic alteration of cell lines (P < 0.05). In conclusion, IRTKS plays an important role in PC tumorigenesis via PI3K/AKT pathway phosphorylated activation, and has a potential clinical application value in prognosis for PC.

The Antitumor Activity of hAMSCs Secretome in HT-29 Colon Cancer Cells Through Downregulation of EGFR/c-Src/IRTKS Expression and p38/ERK1/2 Phosphorylation.

Colon cancer is considered as one of the main causes of mortality worldwide. Identifying a novel and more effective platform with fewer side effects is still progress. In various cancer types, Epidermal growth factor receptor (EGFR) and c-Src (a key mediator in EGFR signaling pathway) are the key targets for cancer therapy. Moreover, insulin receptor tyrosine kinase substrate (IRTKS or BAI1-associated protein 2-like 1: BAIAP2L1) is a member of the subfamily of inverse BAR (I-BAR) domain proteins, which mediates cell morphology and movement through regulation of actin polymerization. In this study, we employed a co-culture system using Transwell six-well plates. After 72 h, hAMSCs-treated HT-29 cells, EGFR, c-Src, IRTKS, p38, and ERK1/2 expression were analyzed using quantitative real time PCR (qRT-PCR) and western blot methods. The significant reduction in tumor cell growth and motility through downregulation of EGFR/c-Src/IRTKS expression and p38/ERK1/2 phosphorylation in HT-29 cells was demonstrated based on 2D and 3D cell culture models. The induction of cellular apoptosis was also found. Our results support the idea that the hAMSCS secretome has therapeutic effects on cancer cells. However, further experiments will be required to identify the exact molecular mechanisms.

Upregulated circRNA_102231 promotes gastric cancer progression and its clinical significance.

Circular RNAs (circRNAs) are a type of endogenous non-coding RNAs implicated in cancer progression. This study explored the expression levels, clinical implication and possible molecular mechanism of circRNA_102231 in gastric cancer (GC). Gene Expression Omnibus (GEO) was used to analyze differentially expressed circRNAs. CircRNA_102231 expression was verified by qRT-PCR in GC tissues and plasma. The effects of circRNA_102231 was tested by CCK-8, colony formation, EdU and Transwell assays and xenograft tumor model. RNA pull-down and immunoprecipitation (RIP) assays were used to analyze the interaction between circRNA_102231 and IRTKS. CircRNA_102231 expression was significantly upregulated in GC tissue and plasma samples, which can be used as a biomarker for GC diagnosis and prognosis. The function assays showed that circRNA_102231 knockdown inhibited GC cell proliferation and invasion both in vitro and in vivo. CircRNA_102231 was able to bind to IRTKS, increasing IRTKS protein stability, leading to GC progression. Overexpression of IRTKS effectively rescued the reduced cell viability and invasion caused by silencing of circRNA_102231. In sum, our data demonstrate that circRNA_102231 is a novel oncogene in GC and acts as a potential biomarker and therapeutic target for GC patients.AbbreviationscircRNAs: circular RNAs; GC: gastric cancer; GEO: Gene Expression Omnibus; RIP: RNA immunoprecipitation; DEGs: differentially expressed genes.

Upregulated insulin receptor tyrosine kinase substrate promotes the proliferation of colorectal cancer cells via the bFGF/AKT signaling pathway.

BACKGROUND: Some recent studies on insulin receptor tyrosine kinase substrate (IRTKS) have focused more on its functions in diseases. However, there is a lack of research on the role of IRTKS in carcinomas and its mechanism remains ambiguous. In this study, we aimed to clarify the role and mechanism of IRTKS in the carcinogenesis of colorectal cancer (CRC). METHODS: We analysed the expression of IRTKS in CRC tissues and normal tissues by researching public databases. Cancer tissues and adjacent tissues of 67 CRC patients who had undergone radical resection were collected from our center. Quantitative real-time polymerase chain reaction and immunohistochemistry were performed in 52 and 15 pairs of samples, respectively. In vitro and in vivo experiments were conducted to observe the effect of IRTKS on CRC cells. Gene Set Enrichment Analysis and Metascape platforms were used for functional annotation and enrichment analysis. We detected the protein kinase B (AKT) phosphorylation and cell viability of SW480 transfected with small interfering RNAs (siRNAs) with or without basic fibroblast growth factor (bFGF) through immunoblotting and proliferation assays. RESULTS: The expression of IRTKS in CRC tissues was higher than that in adjacent tissues and normal tissues (all P < 0.05). Disease-free survival of patients with high expression was shorter. Overexpression of IRTKS significantly increased the proliferation rate of CRC cells in vitro and the number of tumor xenografts in vivo. The phosphorylation level of AKT in CRC cells transfected with pLVX-IRTKS was higher than that in the control group. Furthermore, siRNA-IRTKS significantly decreased the proliferation rate of tumor cells and the phosphorylation level of AKT induced by bFGF. CONCLUSION: IRTKS mediated the bFGF-induced cell proliferation through the phosphorylation of AKT in CRC cells, which may contribute to tumorigenicity in vivo.

Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector.

Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.

Insulin receptor tyrosine kinase substrate activates EGFR/ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma.

Insulin receptor tyrosine kinase substrate (IRTKS) is closely associated with actin remodelling and membrane protrusion, but its role in the pathogenesis of malignant tumours, including hepatocellular carcinoma (HCC), is still unknown. In this study, we showed that IRTKS was frequently upregulated in HCC samples, and its expression level was significantly associated with tumour size. Enforced expression of IRTKS in human HCC cell lines significantly promoted their proliferation and colony formation in vitro, and their capacity to develop tumour xenografts in vivo, whereas knockdown of IRTKS resulted in the opposite effects. Furthermore, the bromodeoxyuridine (BrdU) incorporation analyses and propidium iodide staining indicated that IRTKS can promote the entry into S phase of cell cycle progression. Significantly, IRTKS can interact with epidermal growth factor receptor (EGFR), results in the phosphorylation of extracellular signal-regulated kinase (ERK). By contrast, inhibition of ERK activation can attenuate the effects of IRTKS overexpression on cellular proliferation. Taken together, these data demonstrate that IRTKS promotes the proliferation of HCC cells by enhancing EGFR-ERK signalling pathway.

Possible role of IRTKS in Tks5-driven osteoclast fusion.

Podosomes and invadopodia seen in osteoclasts and cancer cells, respectively, are actin-rich membrane protrusions. We recently demonstrated that an adaptor protein, Tks5, which is an established regulator of invadopodia in cancer cells, drives osteoclast-osteoclast fusion as well as osteoclast-cancer cell fusion by generating circumferential podosomes/invadopodia. This finding revealed an unexpected potential of podosomes/invadopodia to act as fusion-competent protrusions. Fusion of biological membranes involves the intricate orchestration of various proteins and lipids. Recent literature suggests the importance of membrane curvature formation in lipid bilayer fusion. In this study, we investigated the expression of Bin-Amphiphysin-Rvs161/167 (BAR) domain superfamily proteins, which have membrane deforming activity, during osteoclastogenesis. We found that IRTKS was specifically induced during osteoclast fusion and interacted with Tks5, suggesting the role of IRTKS in the formation of fusion-competent protrusions via its BAR domain.