Receptor For Activated C Kinase 1 Overexpression Research Articles

Deciphering the neuroprotective mechanisms of RACK1 in cerebral ischemia-reperfusion injury: Pioneering insights into mitochondrial autophagy and the PINK1/Parkin axis.

Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. Invitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. Invivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. Invitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. Invivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.

RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice.

CD4+ T cells are central mediators of protective immunity to blood-stage malaria, particularly for their capacity in orchestrating germinal center reaction and generating parasite-specific high-affinity antibodies. T follicular helper (Tfh) cells are predominant CD4+ effector T cell subset implicated in these processes, yet the factors and detailed mechanisms that assist Tfh cell development and function during Plasmodium infection are largely undefined. Here we provide evidence that receptor for activated C kinase 1 (RACK1), an adaptor protein of various intracellular signals, is not only important for CD4+ T cell expansion as previously implied but also plays a prominent role in Tfh cell differentiation and function during blood-stage Plasmodium yoelii 17XNL infection. Consequently, RACK1 in CD4+ T cells contributes significantly to germinal center formation, parasite-specific IgG production, and host resistance to the infection. Mechanistic exploration detects specific interaction of RACK1 with STAT3 in P. yoelii 17XNL-responsive CD4+ T cells, ablation of RACK1 leads to defective STAT3 phosphorylation, accompanied by substantially lower amount of STAT3 protein in CD4+ T cells, whereas retroviral overexpression of RACK1 or STAT3 in RACK1-deficient CD4+ T cells greatly restores STAT3 activity and Bcl-6 expression under the Tfh polarization condition. Further analyses suggest RACK1 positively regulates STAT3 stability by inhibiting the ubiquitin-proteasomal degradation process, thus promoting optimal STAT3 activity and Bcl-6 induction during Tfh cell differentiation. These findings uncover a novel mechanism by which RACK1 participates in posttranslational regulation of STAT3, Tfh cell differentiation, and subsequent development of anti-Plasmodium humoral immunity.

Receptor for Activated C Kinase 1 counteracts ABSCISIC ACID INSENSITIVE5-mediated inhibition of seed germination and post-germinative growth in Arabidopsis.

ABSCISIC ACID INSENSITIVE5 (ABI5), a key regulator of the abscisic acid (ABA) signalling pathway, plays a fundamental role in seed germination and post-germinative development. However, the molecular mechanism underlying the repression function of ABI5 remains to be elucidated. In this study, we demonstrate that the conserved eukaryotic WD40 repeat protein Receptor for Activated C Kinase 1 (RACK1) is a novel negative regulator of ABI5 in Arabidopsis. The RACK1 loss-of-function mutant is hypersensitive to ABA, while this phenotype is rescued by a mutation in ABI5. Moreover, overexpression of RACK1 suppresses ABI5 transcriptional activation activity for ABI5-targeted genes. RACK1 may also physically interact with ABI5 and facilitate its degradation. Furthermore, we found that RACK1 and the two substrate receptors CUL4-based E3 ligases (DWA1 and DWA2) function together to mediate the turnover of ABI5, thereby efficiently reducing ABA signalling in seed germination and post-germinative growth. In addition, molecular analyses demonstrated that ABI5 may bind to the promoter of RACK1 to repress its expression. Collectively, our findings suggest that RACK1 and ABI5 might form a feedback loop to regulate the homeostasis of ABA signalling in acute seed germination and early plant development.

RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

Pasteurella multocida is a gram-negative bacterium that causes serious diseases in a wide range of animal species. Inflammasomes are intracellular multimolecular protein complexes that play a critical role in host defence against microbial infection. Our previous study showed that bovine P. multocida type A (PmCQ2) infection induces NLRP3 inflammasome activation. However, the exact mechanism underlying PmCQ2-induced NLRP3 inflammasome activation is not clear. Here, we show that NLRP3 inflammasome activation is positively regulated by a scaffold protein called receptor for activated C kinase 1 (RACK1). This study shows that RACK1 expression was downregulated by PmCQ2 infection in primary mouse peritoneal macrophages and mouse tissues, and overexpression of RACK1 prevented PmCQ2-induced cell death and reduced the numbers of adherent and invasive PmCQ2, indicating a modulatory role of RACK1 in the cell death that is induced by P. multocida infection. Next, RACK1 knockdown by siRNA significantly attenuated PmCQ2-induced NLRP3 inflammasome activation, which was accompanied by a reduction in the protein expression of interleukin (IL)-1β, pro-IL-1β, caspase-1 and NLRP3 as well as the formation of ASC specks, while RACK1 overexpression by pcDNA3.1-RACK1 plasmid transfection significantly promoted PmCQ2-induced NLRP3 inflammasome activation; these results showed that RACK1 is essential for NLRP3 inflammasome activation. Furthermore, RACK1 knockdown decreased PmCQ2-induced NF-κB activation, but RACK1 overexpression had the opposite effect. In addition, the immunofluorescence staining and immunoprecipitation results showed that RACK1 colocalized with NLRP3 and that NEK7 and interacted with these proteins. However, inhibition of potassium efflux significantly attenuated the RACK1-NLRP3-NEK7 interaction. Our study demonstrated that RACK1 plays an important role in promoting NLRP3 inflammasome activation by regulating NF-κB and promoting NLRP3 inflammasome assembly.

Arabidopsis thaliana and Oryza sativa receptor for activated C kinase 1 (RACK1) mediated signaling pathway shows hypersensitivity to oxidative stress

Scaffold proteins regulate cellular mechanisms and phenotypic patterns through protein-to-protein interactions. Receptor for Activated C Kinase 1 (RACK1) is a highly conserved protein containing 40 amino acid repeats of tryptophan-aspartic acid dipeptides (WD-repeat). RACK1 regulates environmental stress signaling pathways in plants and has important implications for human diseases (e.g., viral infections, cancer) and organ function (e.g., brain, heart, liver). Three isoforms (RACK1A, RACK1B and RACK1C) in plants are associated with drought, salt uptake, UV and oxidative stress. We characterized the regulatory role of RACK1 in Arabidopsis thaliana and Oryza sativa using gain of function mutants by T-DNA insertion. Wild type A. thaliana normally displays RACK1 promoter activity (GUS staining) only in leaf tips, but GUS staining after oxidative stress (methyl viologen) was dispersed throughout the leaf and elevated in stomata. Overexpression of RACK1 causes a stressed phenotype and retarded growth in heterozygous O. sativa mutants, while a severely stressed phenotype and failure to thrive (within two weeks of germination) was observed in homozygous mutants. Methyl viologen exposure of leaves in RACK1 gain of function O. sativa decreased chlorophyll content and increased reactive oxygen species. Our findings support previous studies indicating that RACK1 regulates ROS production in O. sativa seedlings. Our results provide important baseline data for future fine-scale characterizations of important developmental regulators imposed by the modulation of RACK1 (and other isoforms). Understanding plant-based food security and human disease pathologies (including viral infections) will inform strategies that aim to reduce the negative impacts of each process to sustain society worldwide.

RACK1 regulates centriole duplication through promoting the activation of polo-like kinase 1 by Aurora A.

Breast cancer gene 1 (BRCA1) contributes to the regulation of centrosome number. We previously identified receptor for activated C kinase 1 (RACK1) as a BRCA1-interacting partner. RACK1, a scaffold protein that interacts with multiple proteins through its seven WD40 domains, directly binds to BRCA1 and localizes to centrosomes. RACK1 knockdown suppresses centriole duplication, whereas RACK1 overexpression causes centriole overduplication in a subset of mammary gland-derived cells. In this study, we showed that RACK1 binds directly to polo-like kinase 1 (PLK1) and Aurora A, and promotes the Aurora A-PLK1 interaction. RACK1 knockdown decreased phosphorylated PLK1 (p-PLK1) levels and the centrosomal localization of Aurora A and p-PLK1 in S phase, whereas RACK1 overexpression increased p-PLK1 level and the centrosomal localization of Aurora A and p-PLK1 in interphase, resulting in an increase of cells with abnormal centriole disengagement. Overexpression of cancer-derived RACK1 variants failed to enhance the Aurora A-PLK1 interaction, PLK1 phosphorylation and the centrosomal localization of p-PLK1. These results suggest that RACK1 functions as a scaffold protein that promotes the activation of PLK1 by Aurora A in order to promote centriole duplication.This article has an associated First Person interview with the first author of the paper.

Downregulated expression of RACK1 results in pancreatic cancer growth and metastasis.

BackgroundThe expression and function of the Receptor for Activated C Kinase 1 (RACK1) in cancer growth and metastasis are confused in different cancers, especially in pancreatic ductal adenocarcinoma (PDAC).MethodsOne-hundred and eighty-two PDAC tissue specimens (95 males and 87 females) including pancreatic cancer tissue and para-carcinoma tissue were collected for analysis between 2005 to 2012. Blood phenotypic parameters using cell count and capillary electrophoresis were investigated. HE staining, real time PCR, Western blot analysis, and soft agar assays were performed to determine the role of RACK1.PurposeIn this study, we aim to determine the specific role of RACK1 in the untility of PDAC.ResultsWe found that RACK1 expression was significantly lower in pancreatic cancer tissue than in para-carcinoma normal pancreatic tissue both in clinic and mice with pancreatic cancer at the early stage. Our results suggested that RACK1 silence could significantly promote cell growth and metastasis of pancreatic cancer cells. But we found that the overexpression of RACK1 has the opposite effect in vitro. In vivo MIAPaca-2 cells overexpressing RACK1, the results demonstrated lower metastatic ability than MIAPaca-2 cells. RACK1 overexpression could decrease the NF-κB transactivation activity of MIAPaca-2 cells, which was consistent with the inhibitory effect of RACK1 overexpression on the pro-migration and pro-invasive target gene of NF-κB, while which could be increased by RACK1 silence. RACK1 silence also enhanced protein expression of pro-migration and pro-invasive NF-κB target genes, which on the contrary, could be reversed by IκBα. Besides, RACK1 expression was significantly associated with lymph node metastasis, vessels metastasis, invasion of nerves as well as TNM staging. The 3-year survival rate of patients with high RACK1 expression was significantly higher than those patients with low RACK1 expression. However, RACK1 expression was not an independent risk factor for of the long-term postoperative survival of patients with pancreatic cancer.ConclusionThe obtained results in our study suggested that the low expression of RACK1 was associated with cancer cell growth and metastasis in pancreatic cancer through the activation of the NF-κB pathway. RACK1 could be a potential therapeutic drug target to pancreatic cancer and metastasis.

RACK1 attenuates RLR antiviral signaling by targeting VISA-TRAF complexes

Virus-induced signaling adaptor (VISA), which mediates the production of type I interferon, is crucial for the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes double-stranded viral RNA and interacts with VISA to mediate antiviral innate immunity. However, the mechanisms underlying RIG/VISA-mediated antiviral regulation remain unclear. In this study, we confirmed that receptor for activated C kinase 1 (RACK1) interacts with VISA and attenuates the RIG/VISA-mediated antiviral innate immune signaling pathway. Overexpression of RACK1 inhibited the interferon-β (IFN-β) promoter; interferon-stimulated response element (ISRE); nuclear factor kappa B (NF-κB) activation; and dimerization of interferon regulatory factor 3 (IRF3) mediated by RIG-I, VISA, and TANK-binding kinase 1 (TBK1). A reduction in RACK1 expression level upon small interfering RNA knockdown increased RIG/VISA-mediated antiviral transduction. Additionally, RACK1 disrupted formation of the VISA-tumor necrosis factor receptor-associated factor 2 (TRAF2), VISA-TRAF3, and VISA-TRAF6 complexes during RIG-I/VISA-mediated signal transduction. Additionally, RACK1 enhanced K48-linked ubiquitination of VISA, attenuated its K63-linked ubiquitination, and decreased VISA-mediated antiviral signal transduction. Together, these results indicate that RACK1 interacts with VISA to repress downstream signaling and downregulates virus-induced IFN-β production in the RIG-I/VISA signaling pathway.

High expression of RACK1 is associated with poor prognosis in patients with pancreatic ductal adenocarcinoma.

Receptor for activated C kinase 1 (RACK1) is associated with certain aspects of cancer biology and signaling pathways, but its function in pancreatic ductal adenocarcinoma (PDAC) remains unknown. In the present study, 157 patients with PDAC were enrolled. RACK1 mRNA and protein expression levels were analyzed in PDAC tissues and matched adjacent noncancerous tissues by reverse transcription-quantitative polymerase chain reaction and western blotting. RACK1 expression levels in paraffin-embedded PDAC tissues were determined by immunohistochemistry. The associations between RACK1 expression and clinical data were evaluated using χ2 analysis. The relationship between RACK1 expression and the survival data of patients was analyzed using Kaplan-Meier and log rank tests. RACK1 mRNA and protein were revealed to be overexpressed in PDAC tumor tissues compared with adjacent noncancerous tissues. RACK1 expression was associated with clinical stage (P=0.001), lymph node invasion (P=0.003) and liver metastasis (P=0.001). Furthermore, patients with PDAC and high RACK1 expression demonstrated shorter overall survival times compared with patients with low RACK1 expression (P=0.002). Multivariate analysis indicated that RACK1 overexpression was an independent prognostic factor for patients with PDAC. Overexpression of RACK1 may contribute to tumor progression, and may be a potential prognostic biomarker for patients with PDAC.

Interaction of the Mineralocorticoid Receptor With RACK1 and Its Role in Aldosterone Signaling.

The mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily of ligand-dependent transcription factors with diverse functions including the biological actions of aldosterone. Identification of the various transcriptional coregulators of MR is essential for understanding the complexity of MR signaling pathways under physiological and pathological conditions. We used a yeast two-hybrid system to find proteins that interact with a full-length MR and found, among other proteins, that MR interacted specifically with receptor for activated C kinase 1 (RACK1), a scaffolding protein. Overexpression of RACK1 using a tetracycline-inducible lentivirus in mouse cortical collecting duct M1 cells stably expressing the rat MR and a Gaussia luciferase gene reporter under a hormone-response element promoter resulted in enhanced agonist-dependent MR transactivation. Knockdown of RACK1 protein expression by short hairpin RNAs led to a significant reduction in MR activation of the reporter gene and the endogenous genes Ctla2α and Psca. We also demonstrated that RACK1 regulation of MR action is mediated through phosphorylation by the PKC-β signaling pathway. MR and RACK1 were coimmunoprecipitated using an MR antibody in male Sprague-Dawley brain tissue and M1-rMR cells, and colocalization in M1-rMR cells and male rat brains was confirmed by immunofluorescence and immunohistochemistry. The scaffolding protein RACK1 is associated with MR under basal and agonist-stimulated conditions and facilitates agonist-stimulated MR actions through PKC-β. These findings indicate that RACK1 is a newly described coactivator of MR.

RACK1 is necessary for the formation of point contacts and regulates axon growth.

Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal scaffolding protein that can interact with multiple signaling molecules concurrently through its seven WD40 repeats. We recently found that RACK1 is localized to mammalian growth cones, prompting an investigation into its role during neural development. Here, we show for the first time that RACK1 localizes to point contacts within mouse cortical growth cones. Point contacts are adhesion sites that link the actin network within growth cones to the extracellular matrix, and are necessary for appropriate axon guidance. Our experiments show that RACK1 is necessary for point contact formation. Brain-derived neurotrophic factor (BDNF) stimulates an increase in point contact density, which was eliminated by RACK1 shRNA or overexpression of a nonphosphorylatable mutant form of RACK1. We also found that axonal growth requires both RACK1 expression and phosphorylation. We have previously shown that the local translation of β-actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on RACK1. Thus, we examined the location of members of the local translation complex relative to point contacts. Indeed, both β-actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation. This implies the novel finding that local translation is regulated at point contacts. Taken together, these data suggest that point contacts are a targeted site of local translation within growth cones, and RACK1 is a critical member of the point contact complex and necessary for appropriate neural development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1038-1056, 2017.

Abstract P100: Interaction of the Mineralocorticoid Receptor with RACK1

The mineralocorticoid receptor (MR) is a multifunctional ligand dependent transcription factor in the steroid receptor superfamily. The MR mediates aldosterone regulation of electrolytes and blood pressure. MR transcriptional co-regulators to influence specific gene transcription. We used a yeast two-hybrid system to find proteins that interact with a full-length MR as bait. Results: Among other proteins, we found a specific interaction of MR with RACK1 (Receptor for Activated C Kinase 1), a scaffolding protein. Overexpression of RACK1 using a tetracycline-inducible lentivirus in mouse cortical collecting duct M1 cells stably expressing a reporter Gaussia luciferase gene under a hormone-response element promoter resulted in enhanced agonist-dependent MR transactivation. Inhibition of RACK1 protein expression by short hairpin RNA led to a significant reduction in MR activation of the reporter gene. RACK1 regulation of MR action is mediated through the PKC-β signaling pathway. MR and RACK1 co-precipitated using a MR antibody in Sprague-Dawley brain tissue and M1 cells and immunofluorescent histochemistry showed MR and RACK1 co-localization in Male Sprague-Dawley brains and M1 cells. Conclusion: The scaffolding protein RACK1 is associated with MR under basal and agonist stimulated conditions and facilitates agonist stimulated MR actions through PKC-β. These findings indicate that RACK1 function as a novel co-activator of MR.

The important role of the receptor for activated C kinase 1 (RACK1) in nasopharyngeal carcinoma progression.

BackgroundThe receptor for activated C kinase 1 (RACK1) is involved in various cancers, but its roles in nasopharyngeal carcinoma (NPC) have not yet been fully elucidated.MethodsInitially, RACK1 expression was analyzed by immunohistochemistry in NPC and normal nasopharyngeal (NP) tissues. It was also detected by qPCR and Western blot in NPC cells. Confocal microscope and immunofluorescence were performed to detect the subcellular compartmentalization of RACK1. Subsequently, after up- or down-regulating RACK1 in NPC cells, cell proliferation and migration/invasion were tested using in vitro assays including MTT, EdU, colony formation, Transwell and Boyden assays. Furthermore, several key molecules were detected by Western blot to explore underlying mechanism. Finally, clinical samples were analyzed to confirm the relationship between RACK1 expression and clinical features.ResultsReceptor for activated C kinase 1 expression was much higher in NPC than NP tissues. And RACK1 was mainly located in the cytoplasm. Overexpression of RACK1 promoted NPC cell proliferation and metastasis/invasion, whereas depletion of this protein suppressed NPC cell proliferation and metastasis/invasion. Mechanistically, RACK1 deprivation obviously suppressed the activation of Akt and FAK, suggesting the PI3K/Akt/FAK pathway as one of functional mechanisms of RACK1 in NPC. Furthermore, clinical sample analysis indicated a positive correlation between in vivo expression of RACK1 with lymph node invasion and clinical stage of NPC.ConclusionOur results demonstrate that RACK1 protein plays an important role in NPC development and progression. The upregulation of RACK1 can promote the proliferation and invasion of NPC by regulating the PI3K/Akt/FAK signal pathway. Thus, this study contributes to the discovery of a potential therapeutic target for NPC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0885-x) contains supplementary material, which is available to authorized users.

Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo

Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. The receptor for activated C-kinase 1 (RACK1) serves as a scaffold protein which is recently found to promote angiogenesis. However, the impact of RACK1 on the vascular endothelial growth factor (VEGF) expression in endothelial cells and subsequent choroidal angiogenesis formation remains to be elucidated. In this study, we found that RACK1 and VEGF expression increased, and reached the peak at 7d in mouse CNV model by laser application. Furthermore, on RPE/choroid cryosections, RACK1 co-localized with CD31, suggesting that RACK1 was expressed in endothelial cells. In vitro, RF/6A cell hypoxia model showed that RACK1 expression was up-regulated in parallel with hypoxia-induced factor 1 (HIF-1α) and VEGF expression, reaching the peak at 6h. Silencing of RACK1 suppressed the invasion and tube formation activity of RF/6A cells in ARPE-19 and RF/6A co-culture system, possibly through VEGF signal pathway. Overexpression of RACK1 showed the opposite effect. Intravitreal injection of anti-RACK1 monoclonal antibody predominantly decreased RACK1 and VEGF expression in mouse laser-induced CNV model. Meanwhile, anti-RACK1 monoclonal antibody intravitreal injection also decreased incidence of CNV and leakage area. These data indicated that RACK1 promoted CNV formation via VEGF pathway. Additionally, anti-RACK1 monoclonal antibody significantly decreased CNV in mouse model and may have therapeutic potential in human CNV.

RACK1 overexpression is linked to acquired imatinib resistance in gastrointestinal stromal tumor.

Although treatment with imatinib, which inhibits KIT and PDGFR, controls advanced disease in about 80% of gastrointestinal stromal tumor (GIST) patients, resistance to imatinib often develops. RACK1 (Receptor for Activated C Kinase 1) is a ribosomal protein that contributes to tumor progression by affecting proliferation, apoptosis, angiogenesis, and migration. Here, we found that c-KIT binds to RACK1 and increases proteasome-mediated RACK1 degradation. Imatinib treatment inhibits c-KIT activity and prevents RACK1 degradation, and RACK1 is upregulated in imatinib-resistant GIST cells compared to non-resistant parental cells. Moreover, Erk and Akt signaling were reactivated by imatinib in resistant GIST cells. RACK1 functioned as a scaffold protein and mediated Erk and Akt reactivation after imatinib treatment, thereby promoting GIST cell survival even in the presence of imatinib. Combined inhibition of KIT and RACK1 inhibited growth in imatinib-resistant GIST cell lines and reduced tumor relapse in GIST xenografts. These findings provide new insight into the role of RACK1 in imatinib resistance in GIST.

Downregulation of RACK1 is associated with cardiomyocyte apoptosis after myocardial ischemia/reperfusion injury in adult rats.

The receptor for activated C kinase 1 (RACK1) is a multifaceted scaffolding protein that mediates the shuttling of activated protein kinase C (PKC) to cellular membranes. In addition, RACK1 could decrease cell apoptosis in a variety of disease models. However, the function of RACK1 in cardiomyocyte apoptosis after myocardial ischemia/reperfusion (I/R) is unknown. In this study, male Sprague-Dawley rats were anesthetized and subjected to myocardial I/R insult consisting of 30 min left anterior descending coronary artery (LAD) occlusion followed by reperfusion for 1, 2, 4, 6, 8, 12, and 24 h. The expression of RACK1 was decreased after myocardial I/R and was associated with cardiomyocyte apoptosis. To further verify the relationship between RACK1 and cardiomyocyte apoptosis, H9c2 cardiomyocytes were cultured under hypoxia for 6 h, then maintained in the regular incubator to reoxygenation. After H9c2 cells were transfected with Flag-RACK1 to overexpress RACK1, RACK1 expression was upregulated in hypoxia/reoxygenation (H/R) 4 h group accompanied with the decrease of cleaved caspase-3 and the increase of Bcl-2 expression. Terminal transferase-mediated biotin dUTP nick end labeling (TUNEL) assay showed that RACK1 overexpression inhibited H9c2 cell apoptosis induced by H/R treatment. Our data suggested that RACK1 might suppress cardiomyocyte apoptosis after I/R, providing a novel molecular target for the therapy of ischemia heart disease.

RACK1 expression contributes to JNK activity, but JNK activity does not enhance RACK1 expression in hepatocellular carcinoma SMMC-7721 cells.

Receptor for activated C kinase 1 (RACK1) is up-regulated in hepatocellular carcinoma (HCC) and has been reported to augment c-Jun N-terminal protein kinase (JNK) activity in HCC SMMC-7721 cells. By contrast, activator protein-1, a downstream JNK transcription factor, has been revealed to mediate the overexpression of RACK1 in melanoma cells. Therefore, the association between RACK1 and JNK activity in HCC cells has yet to be completely elucidated. The present study analyzed the effects of RACK1 or JNK loss of function on the levels of RACK1 protein, JNK activity, cell proliferation and apoptosis induced by tumor necrosis factor-related apoptosis inducing ligand in HCC SMMC-7721 cells. It was found that JNK loss of function exhibited no effect on RACK1 expression, whereas a loss of RACK1 function led to reduced JNK activity in SMMC-7721 cells. RACK1 and JNK loss of function resulted in the impaired oncogenic growth of SMMC-7721 cells. The present data further support a pivotal role of RACK1 in mediating enhanced JNK activity in HCC cells and also indicate that a novel mechanism exists for RACK1 overexpression in HCC SMMC-7721 cells.

Receptor for Activated C Kinase 1 (RACK1) Inhibits Function of Transient Receptor Potential (TRP)-type Channel Pkd2L1 through Physical Interaction

Pkd2L1 (also called TRPP3) is a non-selective cation channel permeable to Ca(2+), Na(+), and K(+) and is activated by Ca(2+). It is also part of an acid-triggered off-response cation channel complex. We previously reported roles of the Pkd2L1 C-terminal fragments in its channel function, but the role of the N terminus remains unclear. Using a yeast two-hybrid screening, we found that the Pkd2L1 N terminus interacts with the receptor for activated C kinase 1 (RACK1), a scaffolding/anchoring protein implicated in various cellular functions. This interaction requires the last two Trp-Asp (WD) motifs of RACK1 and fragment Ala(19)-Pro(45) of Pkd2L1. The interaction was confirmed by GST pulldown, blot overlay, and co-immunoprecipitation assays. By (45)Ca tracer uptake and two-microelectrode voltage clamp electrophysiology, we found that in Xenopus oocytes with RACK1 overexpression Pkd2L1 channel activity is abolished or substantially reduced. Combining with oocyte surface biotinylation experiments, we demonstrated that RACK1 inhibits the function of Pkd2L1 channel on the plasma membrane in addition to reducing its total and plasma membrane expression. Overexpressing Pkd2L1 N- or C-terminal fragments as potential blocking peptides for the Pkd2L1-RACK1 interaction, we found that Pkd2L1 N-terminal fragment Met(1)-Pro(45), but not Ile(40)-Ile(97) or C-terminal fragments, abolishes the inhibition of Pkd2L1 channel by overexpressed and oocyte-native RACK1 likely through disrupting the Pkd2L1-RACK1 association. Taken together, our study demonstrated that RACK1 inhibits Pkd2L1 channel function through binding to domain Met(1)-Pro(45) of Pkd2L1. Thus, Pkd2L1 is a novel target channel whose function is regulated by the versatile scaffolding protein RACK1.

RACK1 inhibits morphine re-exposure via inhibition of Src

<title/>Objective: We previously demonstrated that receptor for activated C kinase 1 (RACK1) inhibited phosphorylated extracellular signal-regulated kinase (p-ERK) during morphine reward in mice. In the present study, we examined the role of Src in regulating the inhibition of p-ERK in the brain following RACK1 over-expression during morphine reward.Methods: Mice were subcutaneously injected with morphine on days 2, 4, 6, and 8 after pre-test (day 1), and saline was delivered the following day. After mice showed place preference, RACK1 over-expression plasmid was administered by intraventricular injection 20 minutes after morphine injection on days 11 and 13. Conditioned place preference (CPP) was measured on days 14, 15, 19, and 20.Results: Chronic morphine injection increased Src and p-ERK expression in cortex and hippocampus, and mice exhibited increased place preference. Intraventricular administration of RACK1 reduced Src and p-ERK levels in cortex and hippocampus, as well as morphine reward. At 7 days of final RACK1 administration, the effects of RACK1 on Src and p-ERK disappeared, and morphine place preference was restored.Conclusion: We demonstrated that RACK1 acts on ERK activation via Src in morphine reward in mice.

Receptor for Activated C-Kinase 1 Regulates the Cell Surface Expression and Function of ATP Binding Cassette G2

In a previous report, we identified the receptor for activated C-kinase 1 (RACK1) as a positive regulator of the cellular localization and expression of ATP-binding cassette B4, a phosphatidylcholine translocator expressed on the bile canalicular membrane. In the present study, we focused on the role of RACK1 on ATP-binding cassette G2 (ABCG2), which is responsible for the cellular extrusion of compounds including antitumor drugs. Protein expression of ABCG2 was up-regulated by RACK1 overexpression, although mRNA expression of ABCG2 was not dependent on RACK1. The effect of RACK1 on the expression of ABCG2 on the cell surface was confirmed by the uptake of [(3)H]estrone sulfate, an ABCG2 substrate, into isolated membrane vesicles. The expression of RACK1 affected cellular resistance to mitoxantrone, an anticancer drug excreted by ABCG2, and this effect of RACK1 was abolished in the presence of fumitremorgin C, a selective ABCG2 inhibitor. These results suggest that RACK1 has functional significance as a regulatory cofactor of ABCG2 and is indispensable for the cell surface expression and excretion function of ABCG2. The precise mechanism for RACK1-dependent expression of ABCG2 remains to be clarified, because the results of N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and chloroquine treatment and those of metabolic labeling experiments did not give us clear evidence whether the reduction of ABCG2 expression in RACK1-knocked down cells may be caused by the suppression of ABCG2 protein synthesis or by acceleration of its degradation.