Phosphotyrosine Interaction Domain Research Articles

IGF2BP2 Induces U251 Glioblastoma Cell Chemoresistance by Inhibiting FOXO1-Mediated PID1 Expression Through Stabilizing lncRNA DANCR.

Glioma is the most common type of malignant tumor of the nervous system and is characterized by high mortality and poor outcome. This study aims to investigate the mechanism underlying IGF2 mRNA-binding protein 2 (IGF2BP2) and long noncoding RNA DANCR in etoposide resistance of glioblastoma (GBM) cells. Bioinformatics analysis identified the IGF2BP2-related regulators and DANCR target genes, which were subsequently evaluated by RNA pull-down and RIP assays. We exposed GBM cells to etoposide and thus established etoposide-resistant cells. Through functional experiments, we evaluated the interrelationship among IGF2BP2, DANCR, phosphotyrosine interaction domain containing 1 (PID1), and forkhead box protein O1 (FOXO1) and further assessed their impact on the sensitivity of GBM cells to etoposide. IGF2BP2 and DANCR were highly expressed in glioma cells and tissues, whereas PID1 and FOXO1 were poorly expressed. Mechanistically, overexpression of IGF2BP2 promoted DANCR stability and reduced DANCR methylation, whereas silencing of IGF2BP2 reduced survival of GBM cells and etoposide-resistant cells. Besides, DANCR interacted with FOXO1 to promote the ubiquitination of FOXO1. FOXO1 promoted the transcriptional expression of PID1, enhancing the chemotherapy sensitivity of GBM cells, but overexpression of PID1 reversed the impact of IGF2BP2. Collectively, IGF2BP2 inhibits PID1 expression through the DANCR/FOXO1 axis, inducing drug resistance in GBM cells, and promoting glioma progression.

PID1 promotes HCC development through regulating TME and inhibiting inflammatory factors induced apoptosis

Abstract Objective: PID1 (Phosphotyrosine interaction domain containing 1), an obesity related gene, was reported to induce insulin resistance and play a vital role in lipid metabolism in liver. However, its influence on HCC (hepatocelluar cancer) have not been full clarified. Here, we aims to investigate the mechanism of PID1 in favoring HCC development. Method: Hydrodynamic injection was applied to overexpress PID1 specifically in liver. Adaptive immunity was evaluated through flow cytometry. Lipids metabolism was determined by metabonomics. Western blotting was utilized to determine the effects of PID1 on PI3K/AKT and RAS/RAF/ERK pathway. Result: Overexpression of PID1 specifically in liver induced HCC development within 16 weeks. The frequency of CD4+ and CD8+ T cells decreased whereas the ratio of Gr1+CD11b+ cells increased in mice bearing PID1 plasmid hydrodynamic injection. Moreover, PID1 induced liver metabolism disorder, resulted in upregulation of TG, TC, NEFA, LDL-C as well as lactate levels, thereby involved in regulating TME (tumor microenvironment) and depressed adaptive immunity. Importantly, PID1 inhibited PI3K/AKT pathway, thus restrained phosphorylation of c-RAF on ser259 site induced by p-AKT and promoted phosphorylation of c-RAF on ser338 site, leading to activation of RAS/RAF/ERK pathway. Activated c-RAF targeted to mitochondria to decrease ROS level, which alleviated toxic effect of inflammatory factors and favored HCC cells survival. Conclusion: Our results demonstrated that PID1 provoked HCC development through regulating TME to depress adaptive immunity and inhibiting apoptosis induced by inflammatory factors.

CSIG-29. STRUCTURAL AND FUNCTIONAL STUDIES OF PID1, A NOVEL GROWTH SUPPRESSOR IN BRAIN TUMORS

Abstract BACKGROUND Brain cancers, including medulloblastomas and gliomas, are a major cause of death in children and adults. We reported that PID1 (Phosphotyrosine Interaction Domain containing 1) is a growth suppressor in medulloblastomas and gliomas (PMID: 24300787). PID1 also enhances the anti-tumor effects of chemotherapy (PMID: 28400607). We are now seeking to better understand the structure and mechanism of PID1, in order to utilize this knowledge to develop innovative PID1-based therapies. METHODS PID1 mutants were expressed in E. coli as MBP fusion proteins, purified and used for MALS analysis. Additional epitope tagged PID1 constructs (HA, V5, tGFP, eGFP) were generated, expressed in mammalian cells and analyzed by western blots, immunoprecipitation, and functional assays. RESULTS We carried out screening and testing of multiple mutants of purified PID1 and characterized them. Experiments in cell culture supported presence of similar findings in mammalian cells. Colony assays in glioma and medulloblastoma cell lines identified a region in PID1 that confers the most robust growth-inhibitory effect. Experiments are underway to further refine the boundaries and characteristics of this growth-inhibitory region. CONCLUSIONS This project, which is focused on better understanding of the structure and function of PID1, is uncovering novel aspects of its molecular function. Insights gained from this work will guide studies to develop innovative PID1-based therapies for gliomas and medulloblastomas.

Overexpression of PID1 reduces high density lipoprotein level and functionality in swine.

Phosphotyrosine interaction domain 1 (PID1), a protein with a phosphotyrosine-binding (PTB) domain, interacts with the lipoprotein receptor-related protein 1 (LRP1) to reduce the insulin sensitivity of adipocyte. Considering the role of LRP1 in lipid metabolism, we investigated the effect of PID1 on the content and biological activities of serum lipoproteins in pigs. PID1-transgenic pigs were genetated by sperm and magnetic nanoparticles-mediated method. The levels of PID1 in PID1-transgenic pig's liver were higher than that in the wild-type pig's liver. We found that serum levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I) were significantly reduced in PID1-transgenic pigs. On the other hand, PID1-transgenic pigs displayed increased non-HDL-C levels. Serum levels of total cholesterol and triglycerides were comparable between the PID1-transgenic and the wild-type pigs. Further, the HDL isolated from PID1-transgenic pigs showed a significant reduction in cholesterol efflux ability. In addition, serum superoxide dismutase activity of PID1-transgenic pigs was also obviously lowered compared with that of wild type pigs. In conclusion, these results suggest that PID1 might be able to adjust HDL-C levels in serum and HDL cholesterol efflux ability.

PID1 alters the antilipolytic action of insulin and increases lipolysis via inhibition of AKT/PKA pathway activation.

PurposeThe aim of this study was to investigate the effect of phosphotyrosine interaction domain containing 1 (PID1) on the insulin-induced activation of the AKT (protein kinase B)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway and lipolysis.MethodsSprague–Dawley rats were fed either chow or a high-fat diet (HFD). The levels of insulin, glycerol, free fatty acids (FFAs) and PID1 mRNA expression were measured in the 2 groups. Furthermore, we examined the role of PID1 in the regulation of the AKT/PKA/HSL cascade and lipolysis in the 3T3-L1 cell line.ResultsAdipose tissue from HFD rats exhibited elevated PID1 expression, which showed a positive correlation with insulin levels and lipolysis. In 3T3-L1 adipocytes, we found that the antilipolytic effect of insulin is mediated by AKT and that phosphorylated AKT results in the promotion of PDE3B expression, the dephosphorylation of PKA and HSL and the suppression of glycerol release. However, overexpression of PID1 and treatment with 1 μM isoproterenol and 100 nM insulin for 24 h resulted in an increased release of glycerol and a noticeable inhibition of AKT phosphorylation, PDE3B expression and the phosphorylation of PKA/HSL in 3T3-L1 cells. In contrast, knockdown of PID1 and treatment with the above reagents inhibited lipolysis and activated the phosphorylation of AKT, which resulted in the dephosphorylation of PKA and HSL.ConclusionsOur findings indicate that PID1 in adipose tissue increases lipolysis by altering the antilipolytic action of insulin. This suggests that PID1 may represent a new therapeutic target to ameliorate adipocyte lipolysis and hence improve insulin sensitivity.

Abstract 3461: PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner

Abstract Background: Our group were first to establish a link between PID1 (Phosphotyrosine Interaction Domain containing 1) and cancer, showing PID1's tumor inhibitory effect in medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor (ATRT) brain tumor cell lines and correlation of higher PID1 mRNA with longer patient survival. PID1 was first identified in 2006 and is associated with obesity-related insulin resistance and mitochondrial dysfunction. Here we present a novel interacting partner of PID1 which phosphorylates and modulates its growth suppressive functions. Methods: In silico predictions, in vitro kinase assay, phosphoproteomic analysis, co-immunoprecipitation, SDS-PAGE, western blotting, flow cytometry. Cells: CHLA-07-BSGBM pediatric glioblastoma, U87 glioblastoma and D283 medulloblastoma cell lines, HEK293FT, primary pooled mouse embryo fibroblasts (MEFs). Results: Using prediction algorithms we found that PID1 was a likely phosphorylation target of a member of the G protein-coupled receptor kinase (GRK) family. In vitro kinase assays coupled with phosphoproteomic analysis confirmed this phosphorylation. Overexpressed HA-PID1 and this GRK could be reciprocally co-immunoprecipitated in 293FT cells and endogenous GRK was co-IPd by endogenous PID1 in CHLA-07-BSGBM. A kinase-dead GRK mutant co-IP'd with PID1 similar to the wildtype GRK indicating that the GRK-PID1 interaction did not require the kinase function of this GRK. Deletion mutants of PID1 differentially bound the GRK in co-IPs in a manner that corresponded to the differential growth inhibition these PID1 deletion mutants exerted in glioblastoma and medulloblastoma cell lines. Functionally, siRNA silencing of this GRK caused apoptosis (annexin-PI positive cells and PARP-1 cleavage) in Pid1wt MEFs suggesting that this GRK mediates survival in MEFs. Apoptosis by the GRK siRNA was mitigated in Pid1ko compared to Pid1wt MEFs, suggesting that PID1 functions downstream of the GRK and negatively regulates this GRK's pro-survival signaling. Importantly, we found similar results in glioblastoma cells using gain/loss of function of the two proteins. Conclusions: We found that PID1 is phosphorylated by a member of the GRK family and interacts with it, affecting this GRK's pro-survival activity. Experiments are now uncovering the molecular ordering of PID1 and this GRK in mediating the growth-suppressive effect of PID1 in brain tumors. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Eslam Nouri Nigjeh, Yang Fu, Ebrahim Zandi, Xiaojiang Chen, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3461.

PID1 in adipocytes modulates whole-body glucose homeostasis

The novel obesity-associated protein Phosphotyrosine Interaction Domain containing 1 (PID1) inhibits insulin-PI3K/Akt signaling pathway and insulin-stimulated glucose uptake in vitro. In this study, we generated fat tissue-specific aP2-PID1 transgenic (aP2-PID1tg) mice and PID1 knockout (PID1−/−) mice to explore how PID1 affects glucose metabolism in vivo. We observed insulin resistance and impaired insulin-PI3K/Akt signaling in aP2-PID1tg mice. Consistent with these data, the PID1−/− mice displayed improved glucose tolerance and insulin sensitivity under chow diet, with increased Akt phosphorylation in white adipose tissue (WAT). We further demonstrated that PID1 could interact with low density lipoprotein receptor-related protein 1 (LRP1) but not the insulin receptor (IR) in adipocytes, and its overexpression could lead to decreased GLUT4 level. Our results thus indentify PID1 as a critical regulator of glucose metabolism in adipocytes.

GAS5 suppresses malignancy of human glioma stem cells via a miR-196a-5p/FOXO1 feedback loop

Glioma stem cells (GSCs) make up highly tumorigenic subpopulations within gliomas, and aberrant expression of GSC genes is a major underlying cause of glioma pathogenesis and treatment failure. The present study characterized the expression and function of long non-coding RNA growth arrest specific 5 (GAS5) in GSCs in order to elucidate the molecular mechanisms by which GAS5 contributes to glioma pathogenesis. We demonstrate that GAS5 suppresses GSC malignancy by binding to miR-196a-5p. miR-196a-5p, an onco-miRNA, stimulates GSC proliferation, migration, and invasion, in addition to reducing levels of apoptosis. miR-196a-5p specifically downregulates the expression of forkhead box protein O1 (FOXO1) by targeting its 3′ untranslated region (3′-UTR). FOXO1 upregulates expression of phosphotyrosine interaction domain containing 1 (PID1), thereby inhibiting GSC tumorigenicity and growth. FOXO1 also upregulates migration and invasion inhibitory protein (MIIP), resulting in attenuation of migration and invasion activities. Interestingly, we also show that FOXO1 promotes GAS5 transcription, thus forminga positive feedback loop. These data provide insights into potential new pathways for GSC molecular therapy and suggest that GAS5 may be an efficacious target for glioma treatments.

PID1 increases chemotherapy-induced apoptosis in medulloblastoma and glioblastoma cells in a manner that involves NF\u03baB

Phosphotyrosine Interaction Domain containing 1 (PID1; NYGGF4) inhibits growth of medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor cell lines. PID1 tumor mRNA levels are highly correlated with longer survival in medulloblastoma and glioma patients, suggesting their tumors may have been more sensitive to therapy. We hypothesized that PID1 sensitizes brain tumors to therapy. We found that PID1 increased the apoptosis induced by cisplatin and etoposide in medulloblastoma and glioblastoma cell lines. PID1 siRNA diminished cisplatin-induced apoptosis, suggesting that PID1 is required for cisplatin-induced apoptosis. Etoposide and cisplatin increased NFκB promoter reporter activity and etoposide induced nuclear translocation of NFκB. Etoposide also increased PID1 promoter reporter activity, PID1 mRNA, and PID1 protein, which were diminished by NFκB inhibitors JSH-23 and Bay117082. However, while cisplatin increased PID1 mRNA, it decreased PID1 protein. This decrease in PID1 protein was mitigated by the proteasome inhibitor, bortezomib, suggesting that cisplatin induced proteasome dependent degradation of PID1. These data demonstrate for the first time that etoposide- and cisplatin-induced apoptosis in medulloblastoma and glioblastoma cell lines is mediated in part by PID1, involves NFκB, and may be regulated by proteasomal degradation. This suggests that PID1 may contribute to responsiveness to chemotherapy.

Role of Phosphotyrosine Interaction Domain Containing 1 in Porcine Intramuscular Preadipocyte Proliferation and Differentiation

ABSTRACTPhosphotyrosine interaction domain containing 1 (PID1), a recently identified gene involved in obesity-associated insulin resistance, plays an important role in fat deposition. However, its effect on porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, the plasmid pcDNA3.1(+)-pPID1 was transfected into porcine intramuscular preadipocytes with Lipofectamine 3000 reagent to over-express porcine PID1 (pPID1). Over-expression of pPID1 significantly promoted porcine intramuscular preadipocyte proliferation. Expression of pPID1 mRNA was significantly increased upon porcine intramuscular preadipocyte differentiation. Indirect fluorescent immunocytochemistry demonstrated that pPID1 protein was localized predominantly in the nucleus of porcine intramuscular preadipocyte. The mRNA levels of peroxisome proliferators-activated receptor γ, CCAAT/enhancer binding protein α and lipoprotein lipase were significantly increased by pPID1 over-expression. Over-expression of pPID1 also led to an increase in lipid accumulation which was detected by Oil Red O staining, and significantly increased the intramuscular triacylglycerol content. These results indicate that pPID1 may play a role in enhancing porcine intramuscular preadipocyte proliferation and differentiation.

Microarray Expression Data Identify DCC as a Candidate Gene for Early Meningioma Progression.

Meningiomas are the most common primary brain tumors bearing in a minority of cases an aggressive phenotype. Although meningiomas are stratified according to their histology and clinical behavior, the underlying molecular genetics predicting aggressiveness are not thoroughly understood. We performed whole transcript expression profiling in 10 grade I and four grade II meningiomas, three of which invaded the brain. Microarray expression analysis identified deleted in colorectal cancer (DCC) as a differentially expressed gene (DEG) enabling us to cluster meningiomas into DCC low expression (3 grade I and 3 grade II tumors), DCC medium expression (2 grade I and 1 grade II tumors), and DCC high expression (5 grade I tumors) groups. Comparison between the DCC low expression and DCC high expression groups resulted in 416 DEGs (p-value < 0.05; fold change > 2). The most significantly downregulated genes in the DCC low expression group comprised DCC, phosphodiesterase 1C (PDE1C), calmodulin-dependent 70kDa olfactomedin 2 (OLFM2), glutathione S-transferase mu 5 (GSTM5), phosphotyrosine interaction domain containing 1 (PID1), sema domain, transmembrane domain (TM) and cytoplasmic domain, (semaphorin) 6D (SEMA6D), and indolethylamine N-methyltransferase (INMT). The most significantly upregulated genes comprised chromosome 5 open reading frame 63 (C5orf63), homeodomain interacting protein kinase 2 (HIPK2), and basic helix-loop-helix family, member e40 (BHLHE40). Biofunctional analysis identified as predicted top upstream regulators beta-estradiol, TGFB1, Tgf beta complex, LY294002, and dexamethasone and as predicted top regulator effectors NFkB, PIK3R1, and CREBBP. The microarray expression data served also for a comparison between meningiomas from female and male patients and for a comparison between brain invasive and non-invasive meningiomas resulting in a number of significant DEGs and related biofunctions. In conclusion, based on its expression levels, DCC may constitute a valid biomarker to identify those benign meningiomas at risk for progression.

Eukaryotic expression, purification, identification, and tissuedistribution of porcine PID1

Abstract: Phosphotyrosine interaction domain containing 1 (PID1) is a recently discovered gene related to lipid metabolism and may play an important role in fat deposition. In this study, in order to scale up the production of the active recombinant porcine PID1 (pPID1) protein, we reported the expression and purification of a His-tagged version of pPID1 in the yeast Pichia pastoris. The pPID1 cDNA was cloned into the pPICZαA vector and was expressed in methylotrophic yeast (P. pastoris X33) under control of the alcohol oxidase promoter. The intracellularly expressed recombinant protein was purified by Ni-IDA affinity chromatography, yielding over 95% purity and about 1.8 mg/L. The recombinant protein was identified by Western blot. In addition, the tissue distribution of pPID1 protein was investigated, and expression of pPID1 protein was mainly detected in the skeletal muscles and liver. This study provides a simple and efficient method for yielding a large amount of active recombinant pPID1, which can be useful for further study of the pPID1 protein.

CB-19 * PID1, A NEW GROWTH-INHIBITORY GENE, SENSITIZES MEDULLOBLASTOMA AND GLIOMA CELL LINES TO CHEMOTHERAPY

BACKGROUND: Phosphotyrosine Interaction Domain containing protein-1 (PID1) was discovered in 2006. We recently showed that PID1 inhibits growth of brain tumor cell lines and its mRNA level is directly correlated with survival in glioma and medulloblastoma patients (Erdreich-Epstein et al, Clin Cancer Res). The growth-inhibitory effect of PID1 was due to decreased proliferation and increased cell death. METHODS/RESULTS: We hypothesized that PID1 sensitizes brain tumors to therapy, thus accounting for the longer survival in patients with higher PID1 mRNA. Indeed, while cisplatin (10 µg/ml) or transient PID1 overexpression each increased apoptosis of glioma and medulloblastoma cell lines, combining cisplatin with PID1 resulted in markedly higher apoptosis. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis, suggesting that PID1 is partially required for cisplatin-induced apoptosis. Taken together, this suggests that PID1 sensitizes both glioma and medulloblastoma cell lines to cisplatin. Intriguingly, PID1 mRNA increased in response to chemotherapy in a time- and dose-dependent manner in brain tumor cell lines. The chemotherapy-induced increase in PID1 mRNA was blocked by inhibitors of NFkB, suggesting that regulation of PID1 mRNA increase may be an NF-kB-dependent process. Consistent with this, cisplatin increased activity of an NF-kB promoter reporter. Surprisingly, despite the chemotherapy-induced increase in PID1 mRNA, PID1 protein decreased in response to cisplatin, suggesting post-translational modification(s) of PID1 induced by cisplatin. Ongoing work is focusing on the mechanism and role of PID1 in response to chemotherapy, and examining the effect of PID1 on the response of tumors to chemotherapy in vivo. CONCLUSIONS: Our data show that PID1 sensitizes glioma and medulloblastoma cell lines to chemotherapy, possibly explaining the correlation between higher PID1 mRNA and longer patient survival. This suggests also that PID1 mimetics may act as chemotherapy sensitizers in brain tumor treatment.

Abstract 3975: PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy

Abstract BACKGROUND: Phosphotyrosine Interaction Domain containing protein-1 (PID1) is a gene discovered in 2006 in the context of obesity, which we recently showed inhibits growth of brain tumor cell lines and its mRNA level is directly correlated with survival of patients with gliomas and medulloblastomas (Erdreich-Epstein et al, Clin Cancer Res, In Press). PID1 mRNA was lower in more aggressive medulloblastomas and gliomas, and patients whose tumor had higher PID1 mRNA had longer survival. Moreover, overexpression of PID1 markedly decreased 2D colony formation in cell lines of medulloblastomas, GBM and atypical teratoid rhabdoid tumors (ATRT). The growth-inhibitory effect was due to both decreased proliferation and increased cell death. METHODS/RESULTS: COSMIC showed no mutations of PID1 in brain tumors, suggesting that PID1 was not a formal tumor suppressor gene. Therefore, we hypothesized that PID1 may sensitize brain tumor cells to therapy, and thus account for the longer survival in patients with higher PID1 mRNA. Indeed, while both cisplatin (10µg/ml) or transient PID1 overexpression increased apoptosis of glioma and medulloblastoma cell lines (indicated by increased AnnexinV and mitochondrial depolarization), combining cisplatin with PID1 caused a markedly higher apoptosis. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis (AnnexinV), suggesting that PID1 may be required for cisplatin-induced apoptosis. Taken together, this suggests a sensitizing effect of PID1 to chemotherapy in both glioma and medulloblastoma cell lines. Intriguingly, PID1 mRNA and protein both increased in response to etoposide (5µg/ml), vincristine (50ng/ml) or cisplatin (5µg/ml) in a time- and dose-dependent manner in the brain tumor cell lines. This chemotherapy-induced increase in PID1 mRNA was blocked by inhibitors of NFKB, suggesting that regulation of PID1 may be an NFKB-dependent mechanism. Ongoing work seeks to decipher the mechanism of these effects and test them in vivo in mouse models. CONCLUSIONS: Our data suggest that PID1 acts to sensitize glioma and medulloblastoma cells to chemotherapy, possibly explaining the correlation between higher PID1 mRNA and survival in patients. Citation Format: Jingying Xu, Xiuhai Ren, Anthony Tran, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2014-3975

Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

Although constitutive activation of Janus kinase 3 (Jak3) leads to different cancers, the mechanism of trans-molecular regulation of Jak3 activation is not known. Previously we reported that Jak3 interactions with adapter protein p52ShcA (Shc) facilitate mucosal homeostasis. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and Shc and demonstrate the trans-molecular mechanism of regulation of Jak3 activation by Shc. We show that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of Shc where Jak3 directly phosphorylated two tyrosine residues in Src homology 2 (SH2) domain and one tyrosine residue each in calponin homology 1 (CH1) domain and phosphotyrosine interaction domain (PID) of Shc. Direct interactions between mutants of Jak3 and Shc showed that although FERM domain of Jak3 was sufficient for binding to Shc, CH1 and PID domains of Shc were responsible for binding to Jak3. Functionally Jak3 was autophosphorylated under IL-2 stimulation in epithelial cells. However, Shc recruited tyrosine phosphatases SHP2 and PTP1B to Jak3 and thereby dephosphorylated Jak3. Thus we not only characterize Jak3 interaction with Shc, but also demonstrate the molecular mechanism of intracellular regulation of Jak3 activation where Jak3 interactions with Shc acted as regulators of Jak3 dephosphorylation through direct interactions of Shc with both Jak3 and tyrosine phosphatases.

PID1 (NYGGF4), a New Growth-Inhibitory Gene in Embryonal Brain Tumors and Gliomas

We present here the first report of PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) in cancer. PID1 was identified in 2006 as a gene that modulates insulin signaling and mitochondrial function in adipocytes and muscle cells. Using four independent medulloblastoma datasets, we show that mean PID1 mRNA levels were lower in unfavorable medulloblastomas (groups 3 and 4, and anaplastic histology) compared with favorable medulloblastomas (SHH and WNT groups, and desmoplastic/nodular histology) and with fetal cerebellum. In two large independent glioma datasets, PID1 mRNA was lower in glioblastomas (GBM), the most malignant gliomas, compared with other astrocytomas, oligodendrogliomas and nontumor brains. Neural and proneural GBM subtypes had higher PID1 mRNA compared with classical and mesenchymal GBM. Importantly, overall survival and radiation-free progression-free survival were longer in medulloblastoma patients whose tumors had higher PID1 mRNA (univariate and multivariate analyses). Higher PID1 mRNA also correlated with longer overall survival in patients with glioma and GBM. In cell culture, overexpression of PID1 inhibited colony formation in medulloblastoma, atypical teratoid rhabdoid tumor (ATRT), and GBM cell lines. Increasing PID1 also increased cell death and apoptosis, inhibited proliferation, induced mitochondrial depolaization, and decreased serum-mediated phosphorylation of AKT and ERK in medulloblastoma, ATRT, and/or GBM cell lines, whereas siRNA to PID1 diminished mitochondrial depolarization. These data are the first to link PID1 to cancer and suggest that PID1 may have a tumor inhibitory function in these pediatric and adult brain tumors.

Expression and purification of porcine PID1 gene in Escherichia coli

In this study, in order to scale up the production of recombinant porcine phosphotyrosine interaction domain containing 1 (pPID1), a pET-28a (+)-pPID1 expression plasmid was constructed and transformed into Escherichia coli Rosetta (DE3). The recombinant pPID1 was then purified and identified by western blotting, and was also analyzed in vitro for its function. The recombinant protein was tagged with only a His6 tag at its C-terminus, which could be conveniently purified by affinity column. The protein could be induced for efficient expression with 0.75 mM IPTG for 8 h at 30 °C, yielding approximately 3 mg/L. In vitro biological activity assay demonstrated that the refolded purified recombinant pPID1 increased 3T3-L1 preadipocyte proliferation. This study provides a reliable technique for the recombinant expression and purification of pPID1 proteins.

Porcine phosphotyrosine interaction domain containing 1 modulates 3T3-L1 preadipocyte proliferation and differentiation

The phosphotyrosine interaction domain containing 1 (PID1) gene was firstly isolated from obese subjects and involved in obesity-associated insulin resistance. In the present study, Duroc×Landrace×Yorkshire (DLY) pig PID1 cDNA was cloned. The entire open reading frame of the cloned porcine PID1 is 654 bp. The predicted protein is composed of 217 amino acids residues with a molecular mass of 24,774 Da. Over-expression of porcine PID1 significantly accelerated the proliferation of 3T3-L1 preadipocyte, but inhibited preadipocyte differentiation by decreasing the numerous fat droplets appeared and down-regulating the mRNA expression levels of peroxisome proliferators-activated receptor-γ, CCAAT/enhancer binding protein α, fat acid synthase and lipoprotein lipase. Together, these results suggest that porcine PID1 plays a role in regulating adipose development.

Retraction: Pid1 Induces Insulin Resistance in Both Human and Mouse Skeletal Muscle during Obesity

Obesity is associated with insulin resistance and abnormal peripheral tissue glucose uptake. However, the mechanisms that interfere with insulin signaling and glucose uptake in human skeletal muscle during obesity are not fully characterized. Using microarray, we have identified that the expression of Pid1 gene, which encodes for a protein that contains a phosphotyrosine-interacting domain, is increased in myoblasts established from overweight insulin-resistant individuals. Molecular analysis further validated that both Pid1 mRNA and protein levels are increased in cell culture models of insulin resistance. Consistent with these results, overexpression of phosphotyrosine interaction domain-containing protein 1 (PID1) in human myoblasts resulted in reduced insulin signaling and glucose uptake, whereas knockdown of PID1 enhanced glucose uptake and insulin signaling in human myoblasts and improved the insulin sensitivity following palmitate-, TNF-α-, or myostatin-induced insulin resistance in human myoblasts. Furthermore, the number of mitochondria in myoblasts that ectopically express PID1 was significantly reduced. In addition to overweight humans, we find that Pid1 levels are also increased in all 3 peripheral tissues (liver, skeletal muscle, and adipose tissue) in mouse models of diet-induced obesity and insulin resistance. An in silico search for regulators of Pid1 expression revealed the presence of nuclear factor-κB (NF-κB) binding sites in the Pid1 promoter. Luciferase reporter assays and chromatin immunoprecipitation studies confirmed that NF-κB is sufficient to transcriptionally up-regulate the Pid1 promoter. Furthermore, we find that myostatin up-regulates Pid1 expression via an NF-κB signaling mechanism. Collectively these results indicate that Pid1 is a potent intracellular inhibitor of insulin signaling pathway during obesity in humans and mice.

Abstract 3825: Inhibitory effect of PID1 in medulloblastomas, gliomas and ATRT.

Abstract The recently identified PID1 (Phosphotyrosine Interaction Domain containing 1) is a modulator of insulin signaling in adipocytes and muscle cells. Using a number of independent datasets we show that mean PID1 mRNA was lower in unfavorable medulloblastomas (subgroups 3 and 4) and in glioblastomas compared with favorable medulloblastomas (SHH and WNT subgroups) or normal brain, respectively. Higher PID1 mRNA correlated with longer progression-free survival and overall survival in medulloblastoma patients and with longer overall survival in glioma patients. Ectopic expression of PID1 in medulloblastoma, glioblastoma and ATRT (atypical teratoid rhabdoid tumor) cell lines inhibited colony formation by 68% ± 3.5 (p&amp;lt;0.0001 in total of 24 experiments in triplicates) compared with controls. PID1 effects on apoptosis and proliferation were demonstrated by increased sub-G0/G1 and annexinV staining, decreased S-phase. Additionally, PID1 induced mitochondrial depolarization in the medulloblastoma, glioblastoma, and ATRT cell lines. In keeping with PID1 having a PTB (phosphotyrosine binding) domain, endogenous PID1 co-immunoprecipitated with the low density lipoprotein receptor-related protein-1 (LRP1, which is a potential binding partner for PID1 via its C-terminal NPXY motif). However, PID1 with mutated PTB domain retained its inhibitory effects on colony formation, suggesting that other domains of PID1 mediated (or contributed to) PID1’s inhibitory effect. Analysis of PID1 domains comprising its N-terminal region (D1), C-terminal region (D3) and middle region (D2), each comprising approximately 1/3 of the protein, revealed that indeed, each 1/3 of PID1 inhibited colony formation at least as efficiently as full length PID1. Interestingly, although by itself D2 was more inhibitory than full length PID1, when it was combined with D3, which was also inhibitory by itself, we observed marked mitigation of the inhibitory effect of the combined D2+D5 fragment on colony formation, suggesting functional intramolecular interactions in PID1. These data suggest for the first time that PID1 has a tumor suppressor-like effect in brain tumors and shed light on the molecular mechanism of its inhibitory action. Citation Format: Anat Erdreich-Epstein, Xiuhai Ren, Hong Zhou, Richard Sposto, Lingyun Ji, Stefan Pfister, Marcel Kool, Shahab Asgharzadeh. Inhibitory effect of PID1 in medulloblastomas, gliomas and ATRT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2013-3825