摘要： Inhibition of alpha v beta 3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rob-coupling protein (RCP)-dependent recycling of alpha 5 beta 1 integrin. RCP and alpha 5 beta 1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent alpha 5 beta 1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of alpha 5 beta 1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

关键词： FLNa   IQGAP1   Integrin   Migration   Rac1   RacGAP1  

摘要： Cell migration makes a fundamental contribution to both normal physiology and disease pathogenesis. Integrin engagement with extracellular ligands spatially controls, via the cyclical activation and deactivation of the small GTPase Rac1, the dynamic membrane protrusion and cytoskeletal reorganization events that are required for directional migration. Although the pathways that control integrin-mediated Rac1 activation are reasonably well defined, the mechanisms that are responsible for switching off activity are poorly understood. Here, proteomic analysis of activated integrin-associated complexes suggests filamin-A and IQ-motif-containing GTPase-activating protein 1 (IQGAP1) as candidates that link beta 1 integrin to Rac1. siRNA-mediated knockdown of either filamin-A or IQGAP1 induced high, dysregulated Rac1 activity during cell spreading on fibronectin. Using immunoprecipitation and immunocytochemistry, filamin-A and IQGAP1 were shown to be part of a complex that is recruited to active beta 1 integrin. Mass spectrometric analysis of individual filamin-A, IQGAP1 and Rac1 pull-downs and biochemical analysis, identified RacGAP1 as a novel IQGAP1 binding partner. Further immunoprecipitation and immunocytochemistry analyses demonstrated that RacGAP1 is recruited to IQGAP1 and active beta 1 integrin, and that suppression of RacGAP1 expression triggered elevated Rac1 activity during spreading on fibronectin. Consistent with these findings, reduced expression of filamin-A, IQGAP1 or RacGAP1 triggered unconstrained membrane protrusion and disrupted directional cell migration on fibrillar extracellular matrices. These findings suggest a model whereby integrin engagement, followed by filamin-A, IQGAP1 and RacGAP1 recruitment, deactivates Rac1 to constrain its activity spatially and thereby coordinate directional cell migration.

关键词： 含IQ模序的GTP酶活化蛋白1   肿瘤细胞   增殖  

摘要： 目的探讨含IQ模序的GTP酶活化蛋白1(IQGAP1)对肺癌、乳腺癌和结肠癌细胞增殖的影响。方法转染编码IQGAP1和IQGAP1-C末端的腺病毒载体并检测细胞中IQGAP1和IQGAP1-C的表达水平;转染靶向IQGAP1的小干扰RNA(siRNA),检测内源性IQGAP1的表达水平;用MTT和BrdU掺入法检测IQGAP1对肿瘤细胞增殖的影响;用western blot检测增殖细胞核抗原(PCNA)和细胞周期素E的表达。结果转染腺病毒Ad-IQGAP1或Ad-IQGAP1-C使A549、MCF7和SW480细胞高表达IQGAP1或IQGAP1-C,其增殖能力较Ad-LacZ组均提高(t分别为19.418和25.643、29.283和16.261、23.138和56.739,P均<0.05);A549细胞转染Ad-IQGAP1-C组的促增殖效果明显优于Ad-IQGAP1组(t=25.643,P<0.05);用siRNA沉默内源性IQGAP1的表达后,与对照siRNA组相比,A549、MCF-7和SW480细胞增殖活性受到显著抑制(t分别为18.324、18.931、19.609,P均<0.05)。BrdU掺入法结果显示,增加IQGAP1和IQGAP1-C表达均可明显提高A549、MCF7和SW480的细胞DNA合成率(t分别为14.224和12.079、13.035和15.677、11.291和16.675,P均<0.05),在MCF7和SW480细胞中IQGAP1-C组促DNA合成效果明显优于IQGAP1组(t分别为15.677、16.675,P均<0.05);抑制IQGAP1的表达可显著降低DNA的合成(t分别为8.043、11.381、5.966,P均<0.05);western blot结果显示,在高表达IQGAP1或IQGAP1-C的A549、MCF7和SW480细胞中,PCNA和细胞周期素E表达明显增加(t PCNA分别为6.541和7.460、17.769和13.048、10.261和6.544,P均<0.05;t细胞周期素E分别为17.243和12.258、10.109和14.216、12.011和12.058,P均<0.05);而干扰IQGAP1表达的细胞,与对照siRNA组比较,两者的表达明显降低(t分别为4.475和6.058,7.476和8.404,12.559和14.792,P均<0.05)。结论IQGAP1和IQGAP1-C促进肺癌、乳腺癌和结肠癌细胞增殖,且IQGAP1可能主要通过C末端影响细胞增殖。

关键词： platelet-derived growth factor   IQGAP1   migration   vascular injury   vascular smooth muscle cell  

摘要： Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration and neointimal formation in response to injury. We previously identified IQ-domain GTPase-activating protein 1 (IQGAP1) as a novel VEGF receptor 2 binding scaffold protein involved in endothelial migration. However, its role in VSMC migration and neointimal formation in vivo is unknown. Here we show that PDGF stimulation rapidly promotes IQGAP1 association with PDGF receptor-beta (PDGFR) as well as IQGAP1 tyrosine phosphorylation in cultured VSMC. Overexpression or knockdown of IQGAP1 enhances or inhibits PDGFR autophosphorylation (p-PDGFR), respectively. Immunofluorescence and cell fractionation analysis reveals that PDGF-induced p-PDGFR localized in focal adhesions (FAs), but not caveolae/lipid rafts, is inhibited by IQGAP1 knockdown with siRNA. PDGF stimulation promotes IQGAP1 association with PDGFR/FA signaling protein complex. Functionally, IQGAP1 siRNA inhibits PDGF-induced FA formation as well as VSMC migration induced by PDGF. In vivo, IQGAP1 expression is markedly increased at neointimal VSMC in wire-injured femoral arteries. Mice lacking IQGAP1 exhibit impaired neointimal formation in response to vascular injury. In summary, IQGAP1, through interaction with PDGFR and FA signaling proteins, promotes activation of PDGFR in FAs as well as FA formation, which may contribute to VSMC migration and neointimal formation after injury. Our findings provide insight into IQGAP1 as a potential therapeutic target for vascular migration-related diseases.

关键词： VASCULAR smooth muscle   DISEASES   WOUNDS & injuries   FOCAL adhesions   PLATELET-derived growth factor receptors   PERIPHERAL vascular diseases  

摘要： The article reflects on a Ras GTPase-activating-like protein (IQGAP1) that plays an important role in regulation of Vascular Smooth Muscle Cells (VSMC) migration. It reports that VSMC facilitates by direct interaction between the platelet-derived growth factor receptor (PDGFR) and focal adhesion (FA) proteins in humans. It mentions that PDGF is an acknowledged potent proliferative and pro-migratory stimuli for VSMCs and PDGF concentrations are increased in vascular injury.

关键词： Antioxidants   ERK   Gene Silencing   IQGAP1   MEK   Nrf2   PEITC   Scaffold Proteins  

摘要： Nrf2 plays a critical role in the regulation of cellular oxidative stress. MEK-ERK activation has been shown to be one of the major pathways resulting in the activation of Nrf2 and induction of Nrf2 downstream targets, including phase II detoxifying/antioxidant genes in response to oxidative stress and xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating protein 1), a new Nrf2 interaction partner that we have published previously, was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids 699-905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl isothiocyanate-or MEK-mediated activation of the MEK-ERK-Nrf2 pathway. Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2, which in turn was associated with a decrease in the nuclear translocation of Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes. In the aggregate, these results suggest that IQGAP1 may play an important role in the MEK-ERK-Nrf2 signaling pathway.

关键词： 支架蛋白1   RNA干扰   慢病毒载体  

摘要： 目的制备携带人支架蛋白IQGAP1基因小分子干扰RNA的重组慢病毒。方法分别设计合成针对IQGAP1的4个shRNA慢病毒干扰载体;分别转染293T细胞,利用Western blot验证shRNA的沉默效果,选择其中一个沉默效果较好的shRNA慢病毒干扰载体同源重组产生Lentivirus-IQGAP1-shRNA并测定病毒滴度。结果测序证实,构建携带IQGAP1-shRNA的慢病毒载体具有较好的沉默靶基因的效果,包装慢病毒,病毒滴度为2.0×l010TU/ml。结论成功制备携带IQGAP1-shRNA的重组慢病毒载体并包装出具高效感染力的慢病毒颗粒。

关键词： Cancer   Diabetes   IQGAP1   Metabolism   Signaling  

摘要： Abstract: Epidemiology studies revealed the connection between several types of cancer and type 2 diabetes (T2D) and suggested that T2D is both a symptom and a risk factor of pancreatic cancer. High level of circulating insulin (hyperinsulinemia) in obesity has been implicated in promoting aggressive types of cancers. Insulin resistance, a symptom of T2D, pressures pancreatic β-cells to increase insulin secretion, leading to hyperinsulinemia, which in turn leads to a gradual loss of functional β-cell mass, thus indicating a fine balance and interplay between β-cell function and mass. While the mechanisms of these connections are unclear, the mTORC1-Akt signaling pathway has been implicated in controlling β-cell function and mass, and in mediating the link of cancer and T2D. However, incomplete understating of how the pathway is regulated and how it integrates body metabolism has hindered its efficacy as a clinical target. The IQ motif containing GTPase activating protein 1 (IQGAP1)-Exocyst axis is a growth factor- and nutrient-sensor that couples cell growth and division. Here we discuss how IQGAP1-Exocyst, through differential interactions with Rho-type of small guanosine triphosphatases (GTPases), acts as a rheostat that modulates the mTORC1-Akt and MAPK signals, and integrates β-cell function and mass with insulin signaling, thus providing a molecular mechanism for cancer initiation in diabetes. Delineating this regulatory pathway may have the potential of contributing to optimizing the efficacy and selectivity of future therapies for cancer and diabetes. [Copyright &y& Elsevier]

关键词： Cancer   Diabetes   Signaling   Metabolism   IQGAP1  

摘要： Epidemiology studies revealed the connection between several types of cancer and type 2 diabetes (T2D) and suggested that T2D is both a symptom and a risk factor of pancreatic cancer. High level of circulating insulin (hyperinsulinemia) in obesity has been implicated in promoting aggressive types of cancers. Insulin resistance, a symptom of T2D, pressures pancreatic beta-cells to increase insulin secretion, leading to hyperinsulinemia, which in turn leads to a gradual loss of functional beta-cell mass, thus indicating a fine balance and interplay between beta-cell function and mass. While the mechanisms of these connections are unclear, the mTORC1-Akt signaling pathway has been implicated in controlling beta-cell function and mass, and in mediating the link of cancer and T2D. However, incomplete understating of how the pathway is regulated and how it integrates body metabolism has hindered its efficacy as a clinical target The IQ motif containing GTPase activating protein 1 (IQGAP1)-Exocyst axis is a growth factor- and nutrient-sensor that couples cell growth and division. Here we discuss how IQGAP1-Exocyst, through differential interactions with Rho-type of small guanosine triphosphatases (GTPases), acts as a rheostat that modulates the mTORC1-Akt and MAPK signals, and integrates beta-cell function and mass with insulin signaling, thus providing a molecular mechanism for cancer initiation in diabetes. Delineating this regulatory pathway may have the potential of contributing to optimizing the efficacy and selectivity of future therapies for cancer and diabetes. Published by Elsevier B.V.

关键词： Transcription  

摘要： Aims: Nuclear factor-erythroid-related factor 2 (Nrf2) is a critical transcriptional factor that is used in regulating cellular defense against oxidative stress. This study is aimed at investigating new interacting protein partners of Nrf2 using One-strep tag pull-down coupled with LTQ Orbitrap LC/MS/MS, and at examining the impact on Nr2 signaling by the newly identified IQ motif containing GTPase activating protein 1 (IQGAP1). Results: Using the One-strep tag pull-down and LTQ Orbitrap LC/MS/MS, we identified IQGAP1 as a new Nrf2 interacting partner. Direct interactions between IQGAP1 and Nrf2 proteins were verified using in vitro glutathione S-transferase (GST) pull-down, transcription/translation assays, and in vivo utilizing Nrf2 overexpressing cells. Coexpression of Dsredmono-IQGAP1 and eGFP-Nrf2 increased the stability of eGFP-Nrf2 and enhanced the expression of Nrf2-target gene heme oxygenase-1 (HO-1). To confirm the functional role of IQGAP1 on Nrf2, knock-downed IQGAP1 using siIQGAP1 attenuated the expression of endogenous Nrf2, HO-1 proteins, and Nrf2-target genes GSTpi, GCLC, and NAD(P) H: quinone oxidoreductase 1 (NQO-1). Furthermore, the stability of Nrf2 was dramatically decreased in IQGAP1-deficient mouse embryonic fibroblast (MEF) cells. Since IQGAP1 signaling could be mediated by calcium, treating the cells with calcium showed the translocation of IQGAP1/Nrf2 complex into the nucleus, suggesting that IQGAP1 may play a critical role in Nrf2 stability. Interestingly, consistent with calcium signaling for IQGAP1, treating the cells with calcium functionally enhanced Nrf2-mediated antioxidant responsive element-transcription activity and enhanced the expression of the endogenous Nrf2-target gene HO-1. Innovation: In the aggregate, our current study identifies and functionally characterizes a new Nrf2 partner protein IQGAP1, which may contribute to Nrf2's regulation of antioxidant enzymes such as HO-1. Conclusion: IQGAP1 may play a critical role in the