植物RACK1蛋白研究进展

RACK1（蛋白激酶C受体）是一种色氨酸-天门冬氨酸域（WD40结构）重复蛋白。它是一种多功能支架蛋白, 结合来自不同转导通路的信号分子并在多种哺乳动物发育过程中起关键作用。在植物中也存在RACK1同源基因, 如拟南芥基因组有3个编码RACK1蛋白质的基因, 这3个蛋白质与哺乳动物RACK1在氨基酸水平的相似性都超过75%。此外, 植物RACK1蛋白质包含的WD40数量、位置和蛋白激酶C结合位点的结构域在很大程度上是保守的。该文对植物RACK1蛋白的发现、结构及其在信号转导方面的功能进行综述。     

The anti-inflammatory activity of estrogen in glial cells is regulated by the PKC-anchoring protein RACK-1

It has recently been suggested that estrogen inhibits glial activation and the release of neurotoxic mediators. The mechanisms involved in this anti-inflammatory effect are unclear. We found that an nM concentration of 17-beta estradiol inhibits protein kinaseC-betaII translocation induced by lipopolysaccharide in primary astrocytes. Estradiol treatment did not change the total content of kinaseC-betaII or of lipopolysaccharide receptor, but dose-dependently reduced the levels of receptors for activated C kinases-1 (RACK-1), the anchoring protein involved in protein kinase C (PKC) shuttling. This decrease could thus account for the defective protein kinaseC-betaII activation. Pre-treatment with 1 nmbeta-estradiol, which reduced by approximately 35% the expression of RACK-1, prevented the lipopolysaccharide-induced expression of tumour necrosis factor-alpha mRNA and of the inducible form of nitric oxide (NO) synthase. As a consequence, the production of tumour necrosis factor-alpha and NO were decreased. An antisense oligonucleotide for RACK-1 also reduced tumour necrosis factor-alpha and nitric oxide production on lipopolysaccharide stimulation. These results demonstrate that estrogen reduction of the RACK-1 expression, leading to a defective protein kinase-C activation counteracts the inflammatory response in astrocytes.     

Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro

The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture. Hispidin synthesized in our laboratory to a high degree of purity (checked by 1H and 13C NMR spectroscopy) was shown to be cytotoxic (between 10-3 mol/L and 10-7 mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses (between 10-5 mol/L and 10-7 mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform of protein kinase C (IC50 of 2 × 10-6 mol/L), but not E. coli and placental type XV alkaline phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.     

In vivo glycosylation of MUC1 in airway epithelial cells

The O-glycans that decorate mucin glycoproteins contribute to the biophysical and biochemical properties of these molecules and hence their function as a barrier and lubricant on epithelial surfaces. Alterations in mucin O-glycosylation in certain diseases may contribute to pathology. It is known that both the host cell type and the amino acid sequence of the mucin tandem repeat contribute to the O-glycosylation of a mucin molecule. We expressed an epitope-tagged MUC1 mucin cDNA construct in the airway cell line 16HBE14o- and the colon carcinoma cell line Caco2 and used Fast Atom Bombardment Mass Spectrometry to evaluate the contribution of the host cell to differences in O-glycosylation of a single mucin. Many of the glycans detected on the MUC1 mucin were common to both cell types, as would be predicted from biosynthetic constraints. However, MUC1 synthesized in the airway cell line showed comparatively low levels of sialylation but carried a range of oligo-N-acetyllactosamine structures that were not seen in the colon carcinoma cell line.     

Cloning,expression and genomic structure of a novel human GNB2L1 gene,which encodes a receptor of activated protein kinase C (RACK)*

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.     

The Arabidopsis LECTIN RECEPTOR KINASE-VI.2 is a functional protein kinase and is dispensable for basal resistance to Botrytis cinerea

Sensing of microbial pathogens by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) elicits a defense program known as PAMP-triggered immunity (PTI). Recently, we have shown that the Arabidopsis thaliana L-TYPE LECTIN RECEPTOR KINASE-VI.2 (LecRK-VI.2) positively regulates bacterial PTI. In this report, we suggest by in silico analysis that the kinase domain of LecRK-VI.2 is functional. LecRK-VI.2 also demonstrated auto-phosphorylation activity in vitro in the presence of divalent metal cations indicating that LecRK-VI.2 has the ability to auto-phosphorylate. We further investigate the role of LecRK-VI.2 in Arabidopsis resistance to the necrotrophic fungal pathogen Botrytis cinerea. Disruption of LecRK-VI.2 did not affect Arabidopsis resistance to B. cinerea. Accordingly, wild-type upregulation levels of PTI-responsive WRKY53, FRK1, NHL10, CYP81F2 and CBP60 g after treatment with the fungal PAMP chitin were observed in lecrk-VI.2-1. These data provide evidences that the kinase domain of LecRK-VI.2 is active and show that LecRK-VI.2 is not critical for resistance to the fungal pathogen B. cinerea.     

Molecular and cellular analysis of histamine H1 receptors on cultured smooth muscle cells

Histamine is an important mediator of immediate hypersensitivity for both animals and humans. The action of histamine on target tissues is believed to be mediated by specific cell surface receptors, especially H1 and H2 receptors for hypersensitivity and inflammatory reactions, which involve stimulation of smooth muscle contractility, alterations in vascular permeability, and modifications in the activities of macrophages and lymphocytes. Although the nature of histamine receptors in the brain and peripheral tissues has been studied extensively by many laboratories, the molecular mechanism of histamine receptor-mediated reactions is not fully understood, mainly because histamine receptors are incompletely characterized from the biochemical point of view. In previous studies, we have found that the cultured smooth muscle cell line DDT1MF-2, derived from hamster vas deferens, expresses low-affinity histamine H1 receptors and responds biochemically and functionally to H1-specific stimulation (Mitsuhashi and Payan, J Cell Physiol 134:367, 1988). This cell line provides a model for analyzing the biochemical responses of H1 receptor-mediated reactions in peripheral tissues. In this review, we summarized our recent progress in the study of low-affinity H1 receptors on DDT1MF-2 cells.     

Functional Impairment in Protein Kinase C by RACK1 (Receptor for Activated C Kinase 1) Deficiency in Aged Rat Brain Cortex

Abstract: Several laboratories have reported a lack of protein kinase C (PKC) activation in response to various stimuli in the brain of aged rats. It has been suggested that changes in lipid membrane composition could be related to this functional deficit. However, recent evidence has indicated that the translocation of PKC to the different subcellular compartments is controlled by protein-protein interactions. Recently, a class of proteins, termed receptors for activated C kinase (RACKs), have been described that bind PKC. The present study was conducted to determine whether alterations in RACK1, the best-characterized member of RACKs, were associated with changes in translocation and expression of PKC. Quantitative immunoblotting revealed that RACK1 content was decreased by ∼50% in aged rat brain cortex, compared with that in adult and middle-aged animals. The levels of calcium-independent PKCδ and ε, interacting with RACK1, and related calcium-independent PKC activity were not modified by the aging process. By comparison, phorbol ester-stimulated translocation of this activity and of PKCδ and ε immunoreactivity was absent in cortex from aged animals, as well as the translocation of the calcium-dependent PKCβ, also known to interact with RACK1. These results indicate that a deficit in RACK1 may contribute to the functional impairment in PKC activation observed in aged rat brain.     

TPA induces the expression of EC-SOD in human monocytic THP-1 cells: Involvement of PKC,MEK/ERK and NOX-derived ROS

Abstract

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.     

The tumour suppressor RASSF1A is a novel substrate of PKC

Ras association domain family 1A (RASSF1A) is a tumour suppressor that contains an amino-terminal cysteine-rich region, similar to the diacylglycerol (DAG)-binding domain (C1 domain) found in the protein kinase C (PKC) family of proteins, and a carboxy-terminal Ras-association (RA) domain. In the present study, RASSF1A was identified as a substrate for PKC. Using classical biochemical approaches, it was established that S197 and S203 within the RA domain of RASSF1A are phosphorylated by PKC in vitro and in vivo. Unlike the WT protein, the S197, 203D double mutant of RASSF1A failed to modulate microtubule organization and perinuclear vimentin collapse. By contrast, the equivalent AA mutant of RASSF1A phenocopied the WT protein. These findings indicate that PKC phosphorylation of RASSF1A regulates its ability to reorganize the microtubule network.     

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.     

Isolation and characterization of a human putative receptor protein kinase cDNA STYK1*

Protein kinases (PKs) represent a well studied but most diverse protein superfamily. The covalent, reversible linkage of phosphate to serine, threonine, and tyrosine residues of substrate proteins by protein kinases is probably ubiquitous cellular mechanism for regulation of physiological processes. It is known to us that most signaling pathways impinge at some point on protein kinases. Here we report a human putative receptor protein kinase cDNA STYK1. The STYK1 cDNA is 2749 base pairs in length and contains an open reading frame encoding 422 amino acids. The STYK1 gene is mapped to human chromosome 12p13 and 11 exons were found. RT-PCR showed that STYK1 is widely expressed in human tissues.     

The role of PAK1 in the sensitivity of kidney epithelial cells to ischemia-like conditions

Kidney ischemia, characterized by insufficient supply of oxygen and nutrients to renal epithelial cells, is the main cause of acute kidney injury and an important contributor to mortality world-wide. Earlier research implicated a G-protein coupled receptor (NK1R) in the death of kidney epithelial cells in ischemia-like conditions. P21-associated kinase 1 (PAK1) is involved in signalling by several G-proteins. We explored the consequences of PAK1 inhibition for cell survival under the conditions of reduced glucose and oxygen. Inhibition of PAK1 by RNA interference, expression of a dominant-negative mutant or treatment with small molecule inhibitors greatly reduced the death of cultured kidney epithelial cells. Similar protection was achieved by treating the cells with inhibitors of MEK1, in agreement with the prior reports on PAK1-MEK1 connection. Concomitant inhibition of NK1R and PAK1 offered no better protection than inhibition of NK1R alone, consistent with the two proteins being members of the same pathway. Furthermore, NK1R, PAK and MEK inhibitors reduced the induction of TRAIL in ischemia-like conditions. Considering the emerging role of TRAIL in ischemia-mediated cell death, this phenomenon may contribute to the protective effects of these small molecules. Our findings support further exploration of PAK and MEK inhibitors as possible agents to avert ischemic kidney injury.     

Critical role of X-box binding protein 1 in NADPH oxidase 4-triggered cardiac hypertrophy is mediated by receptor interacting protein kinase 1

NADPH oxidase 4 (NOX4) and the NOX4-related redox signaling are implicated in cardiac hypertrophy. NOX4 is interrelated with endoplasmic reticulum stress (ERS). Spliced X-box binding protein 1 (Xbp1s) is a key mediator of ERS while its role in cardiac hypertrophy is still poorly understood. Recently, receptor interacting protein kinase 1(RIPK1) has been increasingly reported to be associated with ERS. Therefore, we aimed to test the hypothesis that Xbp1s mediates NOX4-triggered cardiac hypertrophy via RIPK1 signaling. In the heart tissue of transverse aortic constriction (TAC) rats and in primary cultured neonatal cardiomyocytes(NCMs) treated with angiotensinII(AngII) or isoproterenol (ISO), NOX4 expression and reactive oxygen species (ROS) generation, and expression of Xbp1s as well as RIPK1-related phosphorylation of P65 subunit of NF-κB were elevated. Gene silencing of NOX4 by specific small interfering RNA (siRNA) significantly blocked the upregulation of NOX4, generation of ROS, splicing of Xbp1 and activation of the RIPK1-related NF-κB signaling, meanwhile attenuated cardiomyocyte hypertrophy. In addition, ROS scavenger (N-acetyl-L-cysteine, NAC) and NOX4 inhibitor GKT137831 reduced ROS generation and alleviated activation of Xbp1 and RIPK1-related NF-κB signaling. Furthermore, splicing of Xbp1 was responsible for the increase in RIPK1 expression in AngII or ISO-treated NCMs. Upregulated RIPK1 in turn activates NF-κB signaling in a kinase activity-independent manner. These findings suggest that Xbp1s plays an important role in NOX4-triggered cardiomyocyte hypertrophy via activating its downstream effector RIPK1, which may prove significant for the development of future therapeutic strategies.     

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1–AMPK axis.     

Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways

In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor α are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCα. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.