P85 Subunit Of PI 3-kinase Research Articles

The C5aR1 complement receptor: A novel immunomodulator of insulin action in skeletal muscle.

The complement system constitutes an integral component of the innate immune system and plays a critical role in adaptive immunity. Activation of this system engenders the production of complement peptide fragments, including C5a, which engage G-protein coupled receptors predominantly expressed in immune-associated cells, such as neutrophils, initiating pro-inflammatory responses. Intriguingly, our investigation has unveiled the presence of C5a receptor 1 (C5aR1) expression within skeletal muscle, a key metabolic tissue and primary target of insulin. Herein, we demonstrate that C5aR1 activation by C5a in differentiated human skeletal muscle cells elicits acute suppression of insulin signalling. This suppression manifests as impaired insulin-dependent association between IRS1 and the p85 subunit of PI3-kinase, a 50% reduction in Akt phosphorylation, and a 60% decline in insulin-stimulated glucose uptake. This impairment in insulin signalling is associated with a three-fold elevation in intramyocellular diacylglycerol (DAG) levels and a two-fold increase in cytosolic calcium content, which promote PKC-mediated IRS1 inhibition via enhanced phosphorylation at IRS1 Ser1101. Significantly, our findings demonstrate that structurally diverse C5aR1 antagonists, along with genetic deletion or stable silencing of C5aR1 by 80% using short-hairpin RNA, effectively attenuate repression of insulin signalling by C5a in LHCN-M2 human skeletal myotubes. These results underscore the potential of heightened C5aR1 activation, characteristic of obesity and chronic inflammatory conditions, to detrimentally impact insulin function within skeletal muscle cells. Additionally, the study suggests that agents targeting the C5a-C5aR axis, originally devised for mitigating complement-dependent inflammatory conditions, may offer therapeutic avenues to ameliorate immune-driven insulin resistance in key peripheral metabolic tissues, including skeletal muscle.

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

Oncogenic Mechanisms of CBL E3 Ubiquitin Ligase Mutations in Myeloid Malignancies

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations are found in a variety of myeloid disorders, including 10-15% of chronic myelomonocytic leukemia (CMML) cases, and specifically disrupt the protein's RING domain, which is responsible for E3 ligase activity; adaptor domains of CBL, including the tyrosine kinase-binding domain (TKB), proline-rich region (PRR) and C-terminal phosphotyrosine (pY) residues, remain intact in the context of RING mutations. In prior studies, CBL RING mutations were associated with hyperactivation of signaling pathways that drive cell proliferation. However, the precise mechanism by which CBL mutants act remains incompletely understood. Here we combined functional assays and mass spectrometry (MS) to comprehensively define the phosphoproteome, CBL interactome and molecular mechanism of signaling hyperactivation in a panel of cell lines expressing an allelic series of CBL RING mutants. We identified the SRC family kinase LYN as a key driver of signaling by CBL RING mutants; furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary CMML patient samples. We generated cell lines expressing wild-type (WT) or RING-mutant CBL using IL3-dependent mouse 32D cells and GM-CSF-dependent human TF1 cells. Cells expressing CBL RING mutants Y371H, C384Y or R420Q had a proliferative advantage over CBL WT or CBL knockout cells. To determine the role of CBL's adaptor domains in the proliferative advantage conferred by CBL RING mutants, we generated double mutants comprising the C384Y RING mutation in cis with mutations in the TKB domain (G306E), PRR (Δ477-688) or pY residues (Y700/731/774F). The proliferative advantage of cells expressing CBL C384Y was significantly reduced with mutation of the TKB domain, PRR or pY residues, indicating that CBL's adaptor domains are critical for the proliferative advantage of cells expressing RING-mutant CBL. To assess the effects of CBL RING mutation on signaling, we used MS to measure global protein phosphorylation in 32D cells expressing CBL WT or CBL C384Y. Activation of LYN and the PI3 kinase (PI3K) pathway were most significantly increased in cells expressing CBL C384Y compared to CBL WT; western blot confirmed increased phosphorylation of LYN, the PI3K p85 subunit and AKT in cells expressing CBL Y371H, C384Y or R420Q. We next employed immunoprecipitation (IP) followed by MS to characterize the global CBL interactome in 32D cells expressing CBL WT or RING mutants Y371H, C384Y or R420Q. In line with the phosphoproteomic analysis, LYN showed significantly increased binding to CBL RING mutants; the PI3K p85 subunit also showed increased binding to CBL RING mutants. Thus, global proteomic analyses revealed that increased binding of LYN and p85 to CBL RING mutants was directly associated with hyperactivation of LYN and PI3K-AKT signaling pathways. Deletion of CBL's PRR reduced interactions with both LYN and p85, and the CBL-p85 interaction required CBL Y731. Genetic ablation or inhibition of LYN by dasatinib decreased binding of p85 to CBL, suggesting that increased CBL Y731 phosphorylation by LYN enabled the CBL-p85 interaction. Indeed, CBL Y731 phosphorylation and AKT activation were diminished by deletion of CBL's PRR, LYN knockout or LYN inhibition by dasatinib. Altogether, these data demonstrated that enhanced LYN activation in cells expressing RING-mutant CBL drives increased CBL phosphorylation, p85 recruitment and downstream AKT signaling. Given the central role of LYN in signaling by CBL RING mutants, we hypothesized that LYN inhibition by dasatinib would abrogate the hyperproliferation of cells expressing CBL RING mutants. Dasatinib blocked the proliferative advantage of 32D and TF1 cells expressing CBL RING mutants. In addition, dasatinib significantly reduced the number of methylcellulose colonies formed by bone marrow mononuclear cells from 2 patients with CBL-mutated CMML; dasatinib treatment of mice xenografted with the same CMML cells resulted in a substantial decrease in leukemia burden compared to vehicle-treated mice. In summary, we have defined a mechanism by which LYN promotes PI3K-AKT signaling through CBL RING mutants. Our data provide rationale for exploring the therapeutic potential of LYN and/or PI3K-AKT inhibition in patients with CBL-mutated myeloid malignancies. Disclosures Ebert: Celgene: Research Funding; Deerfield: Research Funding.

High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells.

Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC) cultures. Retinal EC were cultured in high glucose (HG, 25 mM) for 3 days or peroxynitrite (PN, 100 µM) overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM), or the selective nitration inhibitor epicatechin (100 µM). Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM) controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.

415 - High Glucose-Mediated Tyrosine Nitration Inhibits Survival Signal in Endothelial Cells

Introduction Evidence of retinal oxidative and nitrative stress has been well-documented in models of diabetic retinopathy. Here, we examined the effects of high glucose and peroxynitrite on tyrosine nitration and inhibition of the survival protein PI3-kinase and in particular p85 subunit in retinal endothelial cells. Methods Retinal endothelial cells were cultured in high glucose (25mM) for 3-days or peroxynitrite (100μM) for 1-day in the presence or absence a peroxynitrite decomposition catalyst (FeTPPs, 2.5μM) or the selective nitration inhibitor epicatechin (100μM). Tyrosine nitration of p85 subunit and its association with the p110 subunit were examined using immunoprecipitation. Endothelial cell apoptosis was examined using TUNEL assays and caspase-3 activity. Results High glucose or exogenous peroxynitrite caused significant increase in apoptosis of retinal endothelial cells compared to controls. Treatment with FeTPPs or epicatechin blocked these effects, implying a causal role of peroxynitrite. High glucose- or peroxynitrite-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI 3-kinase that inhibited its association with p110 subunit and inhibition of PI 3-kinase/Akt kinase activity. Inhibiting peroxynitrite formation or blocking tyrosine nitration of p85 restored the activity of PI 3-kinase and Akt kinase and normalized pro-survival function. Conclusions High glucose impairs pro-survival signal and causes accelerated endothelial cell apoptosis via the action of peroxynitrite in causing tyrosine nitration of PI 3-kinase and inhibiting activity of Akt kinase. Inhibitors of nitration can be further developed to combat vascular complication in diabetes.

Role of Herpes simplex virus 1 VP11/12 tyrosine-based binding motifs for Src family kinases, p85, Grb2 and Shc in activation of the phosphoinositide 3-kinase-Akt pathway

The abundant HSV-1 tegument protein VP11/12 encoded by gene UL46 is essential for induction of the PI3K/Akt-signaling pathway during infection. VP11/12 utilizes tyrosine-based motifs within its C-terminal region to bind the SH2 domains of Src family kinases, the p85 subunit of PI3 Kinase and Grb2, and the PTB domain of Shc. We previously proposed that the interaction with SFKs and p85 is used to gain control over the PI3K/Akt signaling pathway. We tested this model by evaluating the effects of mutations that eliminate each of these interactions on the ability of HSV-1 to activate Akt. Inhibiting the interaction of VP11/12 with SFKs, p85 and Grb2 reduced Akt activation, while inhibiting the interaction with Shc had little effect. Overall these data support the suggestion that VP11/12 stimulates the PI3K/Akt pathway by mimicking an activated growth factor receptor.

Antenatal corticosteroids alter insulin signaling pathways in fetal baboon skeletal muscle.

We hypothesize that prenatal exposure to glucocorticoids (GCs) negatively alters the insulin signal transduction pathway and has differing effects on the fetus according to gestational age (GA) at exposure. Twenty-three fetal baboons were delivered from 23 healthy, nondiabetic mothers. Twelve preterm (0.67 GA) and 11 near-term (0.95 GA) baboons were killed immediately after delivery. Half of the pregnant baboons at each gestation received two doses of i.m. betamethasone 24 h apart (170 μg/kg) before delivery, while the other half received no intervention. Vastus lateralis muscle was obtained from postnatal animals to measure the protein content and gene expression of insulin receptor β (IRβ; INSR), IRβ Tyr 1361 phosphorylation (pIRβ), IR substrate 1 (IRS1), IRS1 tyrosine phosphorylation (pIRS1), p85 subunit of PI3-kinase, AKT (protein kinase B), phospho-AKT Ser473 (pAKT), AKT1, AKT2, and glucose transporters (GLUT1 and GLUT4). Skeletal muscle from preterm baboons exposed to GCs had markedly reduced protein content of AKT and AKT1 (respectively, 73 and 72% from 0.67 GA control, P<0.001); IRβ and pIRβ were also decreased (respectively, 94 and 85%, P<0.01) in the muscle of premature GC-exposed fetuses but not in term fetuses. GLUT1 and GLUT4 tended to increase with GC exposure in preterm animals (P=0.09), while GLUT4 increased sixfold in term animals after exposure to GC (P<0.05). In conclusion, exposure to a single course of antenatal GCs during fetal life alters the insulin signaling pathway in fetal muscle in a manner dependent on the stage of gestation.

Abstract W MP77: The Role of Ischemia/Reperfusion Generated Nitrative Stress on VEGF Angiogenic Signaling in Diabetic rats.

Diabetes worsens the outcome and impairs recovery after stroke. We have previously shown that diabetes caused a nitration-dependent dysfunctional cerebral neovascularization via increased vascular endothelial growth factor (VEGF) signalling. Our recent study showed that ischemia/reperfusion (I/R) impaired vascular repair in the post-stroke period in diabetes. Yet, the molecular mechanism is unclear. Our present hypothesis is that I/R-mediated excess peroxynitrite formation impairs VEGF survival and angiogenic signal in a nitration-dependent manner. Methods: Acute ischemic stroke was induced via 90 min middle cerebral artery occlusion in Wistar (Wis) and diabetic Goto-Kakizaki rats (GK). 14 days later brain sections were examined for peroxynitrite generation (nitrotyrosine), survival markers (Akt and P38 activation), and apoptosis markers (caspase-3 and cleaved PARP). Brain micro-vascular endothelial cells (BMVEC) were isolated from Wis and GK rats and subjected to 6 hr hypoxia (0.2% oxygen) followed by 18 h normoxia. Nitrotyrosine, P85 nitration, Akt and P38 activity and caspase-3 cleavage were determined. VEGF signal was assessed via cell proliferation, tube formation and cell migration assay. Results: There was increased basal tyrosine nitration in diabetes. I/R increased nitrotyrosine in control (*2-fold) and diabetic (*3-fold) rats (*p&lt;0.05). This increase was associated with decreased survival signal (20%, *60%) and increased apoptic signal (1.5-fold and *6-fold in Wis and GK, respectively, *p&lt;0.05). In parallel, BMVEC isolated from GK rats showed increased nitrative stress compared to controls (*p&lt;0.05). A second hit by hypoxia/reoxygenation dramatically increased the nitration of P85 subunit of PI3kinase* and activation of p38 MAPK* while decreased Akt activation* (*p&lt;0.05). These effects were associated with impairment of VEGF-induced migration* and restored by treatments with peroxynitrite scavenger FeTPPS* (5 uM) or nitration inhibitor Epicatechin* (200uM) (*p&lt;0.05). Conclusion: Our results demonstrate that I/R diverts prosurvial/angiogenic effects of VEGF to a proapoptotic pathway and suggest peroxynitrite as a novel therapeutic target for improvement of reparative angiogenesis after stroke in diabetes.

Quantitative analysis by surface plasmon resonance of CD28 interaction with cytoplasmic adaptor molecules Grb2, Gads and p85 PI3K

CD28 surface receptors provide co-stimulatory signals that are required for full T cell activation. The CD28 cytoplasmic region has one YMNM and two PXXP motifs as a functional motif. Upon CD28 ligation, Grb2, Gads, and the p85 subunit of PI3 kinase are recruited to the CD28 cytoplasmic region. Here, the interactions between these adaptor proteins and CD28 cytoplasmic domains were analyzed using a Biacore surface plasmon resonance biosensor. For all three adaptor proteins, entire molecules bound more tightly to CD28 than did their isolated SH2 domains. For each adaptor, different outcomes of mutation of CD28’s PXXP motifs on binding affinity indicated that only the SH3 domain of Grb2 bound directly. Regarding binding of SH2s to CD28, the SH2 domains of p85 bound more strongly than those of both Grb2 and Gads. Since intact p85 had a 50-fold higher binding affinity than its fragments, and yet the p85-CD28 interaction does not involve SH3-PXXP binding, binding of both N-terminal and C-terminal SH2s to YMNM may create an “avidity” effect. In contrast, when Grb2 and Gads interact with CD28, binding of their SH3 domains may be important. These results suggest that all these interactions are multivalent, through both SH2 and SH3 domains.

Overexpression Of Leukemia Associated CblY371H Mutation In Transgenic Mice Causes Dosage Dependent Embryonic Lethality

Juvenile Myelomonocytic Leukemia (JMML) is a devastating childhood cancer which is rapidly fatal with infiltration of myeloid cells into multiple organs (Hess, Zutter, Castleberry, & Emanuel, 1996). Based on the observation that uniparental disomy was found in the chromosomal region 11q in JMML patient samples, about 15% of patient samples were found to contain a mutation in c-Cbl (Loh et al., 2009). Moreover, mutant Cbl was also found to be a tumor suppressor gene where a germline mutation results in the predisposition for developing JMML (Niemeyer et al., 2010). The c-Cbl gene encodes a multifunctional adaptor protein which contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif which contain E3 ligase activity, and a C-terminal ubiquitin-associated domain. Previous mutations in myeloid malignancies have been described where mutations occur in the RING finger domain or the linker domain (Caligiuri et al., 2007; Dunbar et al., 2008). Interestingly, a hotspot for mutations at residue 371 exists in JMML patients where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H (Loh et al., 2009). This residue belongs in the linker region of the CBL protein, and it was previously observed that Y371 mediates the binding of c-Cbl to the p85 subunit of PI3 kinase (Blaydes et al., 2001). In vitro, CblY371H mutation does indeed destroy its ligase function resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene was silenced (Niemeyer et al., 2010). However, how mutant Cbl gives rise to JMML and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we tested the oncogenicity of the CblY371H mutation in transgenic mice. Of the 6 founder transgenic mice, we chose L5 line for further analysis because it had the highest level of expression. As expected, overexpression of CblY371H by itself in wild type mice had no phenotype since inactivation of wild type Cbl allele is seen in patients. We, then, generated transgenic mice which were heterozygous for both the BAC transgene and the cbl null mutation (CblY371H; Cbl+/-) and then bred them to Cbl heterozygous knockout mice (Cbl+/-). Because homozygous knockout mice have reduced fertility, we preferentially utilized heterozygous mice for the breeding (El Chami et al., 2005). Interestingly, out of the first 118 progeny genotyped where 1/8 of the pups is expected to be the desired genotype (CblY371H;Cbl-/-), no pups were of the desired genotype suggesting embryonic lethality. Because mice which lack both c-cbl and cbl-b are embryonic lethal, we reasoned that high expression that is achieved from the L5 line was causing a dominant negative affect on cbl-b function resulting in an embryonic lethal phenotype. We then tested if a lower level of the transgene expression would result in viable pups. We used the K5 line which was the second highest expressing transgenic line. We were now able to generate the desired genotype (CblY371H; Cbl-/-) although at less than expected numbers. Thus far, we have generated 4 mice which have the correct genotype which are being monitored for disease. We also monitored the heterozygous mice that carried the transgene (CblY371H; Cbl+/-) which were also asymptomatic without any obvious phenotype. Further studies are being done to characterize the hematopoietic phenotype in these mice by isolating fetal liver cells followed by transplantation into lethally irradiated mice and will be discussed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Role of Herpes Simplex Virus VP11/12 Tyrosine-Based Motifs in Binding and Activation of the Src Family Kinase Lck and Recruitment of p85, Grb2, and Shc

Previous studies have shown that the abundant herpes simplex virus 1 (HSV-1) tegument protein VP11/12, encoded by gene UL46, stimulates phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling: it binds the Src family kinase (SFK) Lck, is tyrosine phosphorylated, recruits the p85 subunit of PI3-kinase, and is essential for the activation of Akt during HSV-1 infection. The C-terminal region of VP11/12 contains tyrosine-based motifs predicted to bind the SH2 domains of SFKs (YETV and YEEI), p85 (YTHM), and Grb2 (YENV) and the phosphotyrosine-binding (PTB) domain of Shc (NPLY). We inactivated each of these motifs in the context of the intact viral genome and examined effects on binding and activation of Lck and recruitment of p85, Grb2, and Shc. Inactivating the p85, Grb2, or Shc motif reduced (p85) or eliminated (Grb2 and Shc) the interaction with the cognate signaling molecule without greatly affecting the other interactions or activation of Lck. Inactivating either SFK motif had only a minor effect on Lck binding and little or no effect on recruitment of p85, Grb2, or Shc. In contrast, inactivation of both SFK motifs severely reduced Lck binding and activation and tyrosine phosphorylation of VP11/12 and reduced (p85) or eliminated (Grb2 and Shc) binding of other signaling proteins. Overall, these data demonstrate the key redundant roles of the VP11/12 SFK-binding motifs in the recruitment and activation of SFKs and indicate that activated SFKs then lead (directly or indirectly) to phosphorylation of the additional motifs involved in recruiting p85, Grb2, and Shc. Thus, VP11/12 appears to mimic an activated growth factor receptor.

Abstract 4301: Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi.

Abstract Gain-of-function mutations of the receptor tyrosine kinase c-Kit are frequently seen in several malignanices with the D816V mutation being the most common. In this study we demonstrate that a mutant of c-Kit/D816V that is deficient in binding of the p85 subunit of PI3-kinase (M724A) has lost its ability to induce ligand-independent cell survival and proliferation while c-Kit/D816V induced PI3-kinases activity was only marginally reduced. Furthermore, inhibitors of PI3-kinase were unable to block c-Kit/D816V-induced cell survival. These data suggest that PI3-kinase plays a role in c-Kit/D816V induced cell transformation which is independent of its lipid kinase activity. By mass spectrometry, we identified a heavily tyrosine-phosphorylated 110 kDa protein associated with p85 to be the p110δ subunit of PI3-kinase and the site of phosphorylation as Tyr 523, and the phosphorylation is not dependent on the lipid kinase activity of PI3-kinase. Knockdown of p110δ in c-Kit/D816V expressing Ba/F3 cells led to reduced cell survival and proliferation while the PI3-kinase activity remained almost intact. The c-Kit/D816V expressing Ba/F3 cells lost their ability to form colonies in semi-solid culture if p110δ was knocked down. Additionally, knockdown of p110δ λεδ τo ρεδυχεδ τυμoρ σιζε, τυμoρ ωειγητ ανδ blood vessel formation ωηεν c-Kit/D816V expressing Ba/F3 cells were subcutaneously injected into nude mice. Taken together, these results suggest a kinase independent role of p110δ in the D816Vmutant of c-Kit mediated cell transformation and suggest that p110δ could potentially be a selective therapeutic target for c-Kit/D816V expressing tumors. Citation Format: Jianmin Sun, Sofie Mohlin, Alicia Lundby, Ulf Hellman, Sven Påhlman, Jesper V. Olsen, Lars Rönnstrand. Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi. [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 4301. doi:10.1158/1538-7445.AM2013-4301

Molecular Mechanism behind Rotavirus NSP1-Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85 Subunit of PI3 Kinase

Our previous study had reported on the interaction of rotavirus NSP1 with cellular phosphoinositide 3-kinase (PI3K) during activation of the PI3K pathway (P. Bagchi et al., J. Virol. 84:6834-6845, 2010). In this study, we have analyzed the molecular mechanism behind this interaction. Results showed that this interaction is direct and that both α and β isomers of the PI3K regulatory subunit p85 and full-length NSP1 are important for this interaction, which results in efficient activation of the PI3K/Akt pathway during rotavirus infection.

Bradykinin‐induced cell migration and COX‐2 production mediated by the bradykinin B1 receptor in glioma cells

Bradykinin is produced and acts at the site of injury and inflammation. Recent reports have also shown that bradykinin selectively modulates blood-tumor barrier permeability. However, the molecular mechanisms and pathologic roles underlying bradykinin-induced glioma migration remain unclear. Glioma is the most common primary adult brain tumor, with a poor prognosis because of the ease with which tumor cells spread to other regions of the brain. In this study, we found that bradykinin increases the cell migration and expression of cyclo-oxygenase-2 (COX-2) in glioma cells. Bradykinin-mediated migration was attenuated by the selective COX-2 inhibitor NS-398. Moreover, increased motility of glioma cells and expression of COX-2 were mimicked by a bradykinin B1 receptor (B1R) agonist and markedly inhibited by a B1R antagonist. Bradykinin-mediated migration was attenuated by phosphoinositide 3-kinase (PI-3 kinase)/AKT inhibitors LY 294002 and wortmannin. Bradykinin stimulation also increased the phosphorylation of the p85 subunit of PI-3 kinase and serine 473 of AKT. Treatment of bradykinin with AP-1 inhibitors Tanshinone IIA and curcumin also reduced COX-2 expression and glioma cell migration. Moreover, treatment of bradykinin also induced phosphorylation of c-Jun in glioma cells. AP-1 promoter analysis in the luciferase reporter construct showed that bradykinin increased AP-1 transcription activity and was inhibited by LY 294002 and wortmannin. One mechanism underlying bradykinin-directed migration is transcriptional up-regulation of COX-2 and activation of the B1R receptor, PI-3 kinase, AKT, c-Jun, and AP-1 pathways.

C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase

In naive T cells, Lck exerts a negative control on the ERK/MAPK pathway. We show that c-mip (c-maf inducing protein) interacts with the p85 subunit of PI3 kinase and inactivates Lck, which results in Erk1/2 and p38 MAPK activation. This effect is not enough to activate AP1 given the inability of ERK to migrate into the nucleus and to transactivate its target genes. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, which blocks the nuclear translocation of ERK1/2. This dual effect of c-mip is unique and might represent a potential mechanism to prevent the development of an immune response. Structured summaryMINT-7383650: p85 (uniprotkb:P27986) physically interacts (MI:0915) with c-Mip (uniprotkb:Q8IY22) by anti bait coimmunoprecipitation (MI:0006)MINT-7383661: c-Mip (uniprotkb:Q8IY22) physically interacts (MI:0915) with p85 (uniprotkb:P27986) by anti tag coimmunoprecipitation (MI:0007)MINT-7383676: p85 (uniprotkb:P27986) physically interacts (MI:0915) with p110 (uniprotkb:P42336) by anti bait coimmunoprecipitation (MI:0006)MINT-7383689, MINT-7383711: Dip-1 (uniprotkb:Q80SY4) physically interacts (MI:0915) with c-Mip (uniprotkb:Q8IY22) by anti tag coimmunoprecipitation (MI:0007)

C-Cbl Regulates Interaction of Immature Hematopoietic Cells with the Bone Marrow Microenvironment by Rac GTPase-Mediated Cytoskeletal Signals.

Abstract 3643Poster Board III-579<Background and purpose>c-Cbl is a ubiquitin E3 ligase and functions as a negative regulator for signals induced by various activated tyrosine kinases, by promoting ubiquitination and proteasomal degradation of these kinases. This inhibitory action is mainly mediated by the tyrosine kinase binding (TKB) and RING finger domains located in the NH3-terminal part of the molecule and these domains are evolutionally well-conserved from nematodes. But mammalian c-Cbl has a stretched structure toward the COOH-terminal end with putative tyrosine residues that can interact with p85 subunit of PI3-kinase, and several reports have denoted that this interaction may be important for cytoskeletal regulation. Recently, mutations of the c-cbl gene have been reported in hematopoietic malignancies, and regulation of hematopoietic stem/progenitor cells (HSPCs) by Cbl is attracting attention. Since it is well-known that cytoskeletal dysregulation is often associated with malignant transformation, in this study, we investigated cytoskeletal regulatory mechanisms mediated by Cbl in hematopoietic cells, using Cbl deficient mice. <Methods and results>To examine the migratory capacity of the HSPCs, lineage negative (Lin(-)) bone marrow cells were set in the Boyden Chamber assay for SDF-1 and fibronectin (FN) were performed. We found that Cbl deficient Lin(-) cells showed significantly decreased migration to these chemoattractants; the migration capacity of the Cbl deficient cells was one eighth for SDF-1 (p = 0.01) and one third for FN (p = 0.007), respectively, compared with the wild-type counterparts. Then, to evaluate in vivo homing ability to the bone marrow microenvironment, transplantation assays were performed. We transplanted 2.5 × 10e6 of Lin(-) HSPCs from Cbl deficient or wild-type Ly5.2 mice into sublethally irradiated wild-type Ly5.1 mice. Three hours after transplantation, chimerism of the transplanted Ly5.2 cells in the bone marrow was examined, and we found that the number of transplanted cells was significantly smaller in Cbl deficient cells, showing that homing capacity of HSPCs in Cbl deficient mice was impaired. Moreover, when we administered G-CSF to Cbl deficient mice, a significantly larger number of Lin(-) Sca1(+) c-Kit(+) cells were mobilized from the bone marrow (p = 0.01), indicating that in Cbl deficient mice, mobilization of HSPCs by G-CSF was also affected. In bone marrow cells or Lin(-) HPSCs of Cbl deficient mice, activity of Rac, a member of small G-protein GTPases, was significantly decreased (p = 0.002). These data ware supported by the experiments with embryonic fibroblast; Rac activities induced by FN stimulation were weaker in Cbl deficient fibroblasts than wild-type ones, and Cbl deficient fibroblasts showed impaired actin rearrangement. <Discussion>We found that Cbl deficient HSPCs showed impaired migration activities to chemoattractants and altered homing and mobilization to and from the bone marrow. Furthermore, Cbl deficient cells had impaired activation of Rac. Currently, little is known about the relationship between Cbl-mediated signals and cytoskeletal regulator Rac, and in this study, we found that Cbl is a positive regulator of Rac activity in the bone marrow hematopoietic cells. Rac activity is reported to be necessary for homing and retention of HSPCs in the bone marrow microenvironment, and our results indicate that interaction of HSPCs with the bone marrow microenvironment, e.g. trafficking of these cells, is regulated by Cbl via Rac GTPase signals. Disclosures:No relevant conflicts of interest to declare.

Abstract 5803: Interaction of Docking Protein Gab1 With Tyrosine Phosphatase SHP2 Regulates Hepatocyte Growth Factor-dependent ERK5 Activation and Upregulation of KLF2 in the Vascular Endothelium

Background : Docking protein Gab1 has been reported to have crucial roles for activation of both mitogen-acitvated protein kinase and AKT downstream of various receptor tyrosine kinases. In the last scientific session, we reported that endothelium-specific Gab1 knockout mice (Gab1ECKO) displayed severe limb necrosis after operatively-induced hindlimb ischemia (HLI) due to the defect of angiogenic response. We also reported that Gab1 was essential for activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) via tyrosine phosphatase SHP2 and AKT via PI3-kinase p85 subunit in human umbilical vein endothelial cells (HUVECs) in response to hepatocyte growth factor (HGF), respectively. Here, we investigated the downstream target of the HGF/Met/Gab1-dependent signaling pathway responsible for the maintenance of endothelium. Methods and Results : We examined the effect of adenovirus-mediated overexpression of β -galactosidase ( β -gal), wild-type Gab1 (Gab1 WT ), or mutated Gab1 which can’t bind with SHP2 (Gab1 ΔSHP2 ) or p85 (Gab1 Δp85 ) on the activation of downstream signaling pathways in HUVECs. HGF-induced activation of both ERK1/2 and ERK5 was enhanced in the HUVECs expressing Gab1 WT or Gab1 Δp85 , but abrogated in those expressing Gab1 ΔSHP2 , compared with control cells expressing β -gal. HGF-induced AKT activation was specifically attenuated in cells expressing Gab1 Δp85 , compared with other three groups. Using DNA microarray analysis, we found that the stimulation with HGF upregulated the expression of Kruppel-like factor 2 (KLF2), a crucial transcriptional factor for endothelial maintenance, and thrombomodulin, a well-known target gene of KLF2, through interaction of Gab1 with SHP2 in HUVECs. Next, we examined the involvement of MEK5-ERK5 pathway for the KLF2 gene expression. HGF-dependent upregulation of KLF2 was almost abrogated by the adenoviral overexpression of either dominant-negative MEK5 or dominant-negative ERK5 in HUVECs. Furthermore, the expression of KLF2 and TM in the endothelium was significantly decreased in the Gab1ECKO, compared with control after HLI. Conclusion : Interaction of Gab1 with SHP2 is essential for both activation of ERK5 and upregulation of KLF2 in response to HGF in the endothelium.

Ligand-induced EpoR internalization is mediated by JAK2 and p85 and is impaired by mutations responsible for primary familial and congenital polycythemia

AbstractEpo-induced endocytosis of EpoR plays important roles in the down-regulation of EpoR signaling and is the primary means that regulates circulating Epo concentrations. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Both JAK2 kinase activity and EpoR cytoplasmic tyrosines are important for ligand-dependent EpoR internalization. Phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain bind p85 subunit of PI3 kinase on Epo stimulation and individually are sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85α and p85β or expression of their dominant-negative forms, but not inhibition of PI3 kinase activity, dramatically impaired EpoR internalization, indicating that p85α and p85β may recruit proteins in the endocytic machinery on Epo stimulation. Furthermore, mutated EpoRs from primary familial and congenital polycythemia (PFCP) patients lacking the 3 important tyrosines do not bind p85 or internalize on stimulation. Addition of residues encompassing Y429 and Y431 to these truncated receptors restored p85β binding and Epo sensitivity. Our results identify a novel PI3 kinase activity-independent function of p85 in EpoR internalization and support a model that defects of internalization in truncated EpoRs from PFCP patients contribute to Epo hypersensitivity and prolonged signaling.

The C3d/Epstein-Barr virus receptor (gp140, CR2, CD21, EBV/C3dR) triggers distinct signaling pathways, independently of CD19 or BCR, in human B lymphocytes. (84.1)

Abstract We originally isolated gp140, the receptor of human C3 (the third component of complement) from Raji, a human B lymphoma cell line (1981). gp140 is the C3d receptor (1983, 1985a) and also the Epstein-Barr virus receptor (1985b). Activated EBV/C3dR regulates B lymphocyte proliferation (1985c). EBV interaction with EBV/C3dR is the first step of B lymphocyte transformation (1985b). Activated EBV/C3dR interacts through its intracytoplasmic domain, with the p53 oncoprotein (1989, 1992, 1995) or a p68 Cabp (1991, 1992, 1995). We identified the two first signaling pathways specifically triggered by activated EBV/C3dR. The first pathway, independent of CD19 and BCR activation, is characterized by: 1) activation of pp60src kinase to tyrosine phosphorylate nucleolin; 2) tyrosine phosphorylated nucleolin interacts with the SH2 domains of the p85 subunit of PI-3 kinase, activating this latter; 3) activated PI 3-kinase recrutes the AKT-GSK3 pathway (1999, 2001, 2003). The second pathway is characterized by: 1) Cbl phosphorylation; 2) interactions of phosphorylated Cbl with Crk-L, Syk and the p85 subunit of PI-3-kinase; 3) dissociation of phosphorylated Cbl from Vav (2006). This dissociation, opposed to the Cbl-Vav association triggered by CD19 or BCR, emphasizes the specificity of EBV/C3dR signaling pathways. We herein identified the functional relationship between these two pathways.

TGFβ inhibits GM‐CSF‐induced phosphorylation of ERK and MEK in human myeloid leukaemia cell lines via inhibition of phosphatidylinositol 3‐kinase (PI3‐k)

Activation of SMAD-independent p44/42 MAPK (ERK1/2) signalling by TGFbeta has been recently reported in various cell types. However, the mechanisms for the linkage between the SMAD-dependent and -independent pathways are poorly understood. In this study, we investigated whether TGF-beta activates the ERK pathway and how TGFbeta communicates with the MAP kinase signals induced by a mitogen, in human myeloid leukaemia cells. TGFbeta dramatically suppressed proliferation of MV4-11 and TF-1 cells without detectable phosphorylation of ERK1/2 and MEK1/2 for the duration of 48 h, as detected by MTT assay and Western blot analysis, respectively. In contrast, GM-CSF induced rapid and transient phosphorylation of MEK1/2 and ERK1/2 and up-regulated cell proliferation. Both GM-CSF-induced ERK1/2 activation and cell proliferation were significantly inhibited by TGFbeta. GM-CSF also induced transient phosphorylation of the p85 subunit of PI3-kinase. Corresponding to this change, phosphorylated p85 was found to bind to the GM-CSF receptor-alpha subunit, as detected by immunoprecipitation and Western blot analysis. PD98059, a selective inhibitor of MEK, blocked GM-CSF-induced phosphorylation of MEK and ERK but not p85. However, TGFbeta and LY294002, a potent inhibitor of PI3-kinase, significantly inhibited phosphorylation of both p85 and ERK1/2. These studies thus indicate that TGFbeta does not activate the ERK pathway but turns off the GM-CSF-induced ERK signal via inhibition of the PI3-kinase-Akt pathway, in these human leukaemia cells.