Biochemical and Biophysical Research Communications
Calcineurin stimulates the expression of inflammatory factors in RAW 264.7 cells by interacting with proteasome subunit alpha type 6
Introduction
Calcineurin (CN), a Ca2+-dependent serine/threonine-specific protein phosphatase, is directly regulated by calcium and calmodulin (CaM). CN consists of a catalytic subunit (CNA, 60 KD) and a regulatory subunit (CNB, 19 KD) [1], [2]. CNA contains a regulatory subunit-binding domain, a CaM-binding domain (CBD) and an auto-inhibitory domain (AI). Deletion mutants lacking the CBD and AI have constitutive phosphatase activity [3]. The key roles of CN have been demonstrated by using the immunosuppressant drugs cyclosporinA (CsA) and tacrolimus (FK506), which from inhibitory complexes by binding to their respective cytoplasmic receptors, cyclophilin and FKBP [4]. CN participates in a variety of physiological and pathological processes, including T-cell activation, apoptosis, ion channel regulation, neurodegenerative disease, and cardiac myocyte hypertrophy [5], [6], [7], [8], and is therefore seen as a regulator of many intracellular signaling events.
Protein degradation is essential for many intracellular processes, and proteasomes play a critical role in degradation. The 26S proteasome, a multi-subunit enzyme complex, is a major cellular non-lysosomal protease [9]. Proteasome subunit alpha type 6 (PSMA6) is one of the alpha-type subunits of the 20S proteasome core complex. It not only participates in the ubiquitin–proteasome pathway (UPP) [10], but also interacts with intracellular proteins such as HIV-1 Tat, PLK1, CUL1, RBX1 and SLX1B, which are implicated in HIV replication, cell proliferation and cell cycle control, tumor growth, apoptosis, stress responses and DNA repair and recombination [11], [12], [13], [14]. Moreover PSMA6 protein is significantly reduced in level in the renal cortex of diabetic rats, and is thought to be involved in the initiation and progression of diabetic nephropathy [15]. Recent studies have shown that elevated levels of PSMA6 increase the risk of myocardial infarction, and inhibition of PSMA6 expression in cultured cells decreases inflammation [16], [17], [18]. These findings have led to increased interest in PSMA6.
Nuclear transcription factor κB (NF-κB) is a major and essential activator that regulates the expression of genes related to inflammation, such as interleukin-6, interleukin-12, interleukin-1β and tumor necrosis factor α [19], [20], [21]. In unstimulated cells, NF-κB activity is inhibited by binding to a family of inhibitors, called IκBs, and activation of NF-κB is initiated by signal-induced degradation of IκB proteins [22]. Degradation occurs primarily via the ubiquitin–proteasome pathway.
In the present study we found that CN stimulated immune responses and NF-κB activation in RAW 264.7 macrophages. We also showed that it increased the proteolytic activity of proteasomes by interacting directly with PSMA6, and stimulated the transcriptional activity of NF-κB via ubiquitin–proteasome pathway-dependent degradation of IκB family proteins.
Section snippets
Materials
Anti-calcineurin (rabbit IgG), anti-IκBα (rabbit IgG) and anti-IκBβ (rabbit IgG) were from Cell Signaling Technology (Massachusetts, USA). Anti-PSMA6 (mouse IgG) and anti-20S proteasome (mouse IgG) were purchased from Santa Cruz Biotechnology (San Diego, USA). Anti-β-actin (mouse IgG) was from Proteintech Group (Chicago, USA) and anti-GST (mouse IgG) and anti-HA were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China).
Cell culture
RAW 264.7 cells were grown in Dulbecco’s modified Eagle’s
CN interacts with proteasome subunit α type 6
The primary aim of our study was to identify new substrates of CN. For this purpose, we performed binding assays with yeast expressing the BD–CN protein and an AD-library. This led to the identification of a cDNA clone encoding the 20S proteasome subunit alpha type 6 (NM_002791.1). Further study showed that CN interacted specifically with that proteasomal subunit (Fig. 1A).
To confirm this interaction, we performed GST pull-down assays using the catalytic subunit of CN, and GST–PSMA6 fusion
Discussion
CN has a much narrower substrate range than other phosphatases, and there has been no previous report of any interaction between CN and proteasomal subunits. In this study, we demonstrate a novel interaction between CN and PSMA6, a subunit of the 20S proteasome. Our findings indicate that PSMA6 interacts with CN both in vivo and in vitro, and that the N-terminal domain of PSMA6 is important for this interaction. Subunits of the proteasome complex interact with many proteins, and a large
Acknowledgment
This work was supported by the National Natural Science Foundation of China.
References (33)
- et al.
Calcineurin is a common target of cyclophilin–cyclosporin A and FKBP–FK506 complexes
Cell
(1991) - et al.
Calcineurin immunoreactivity in Alzheimer’s disease
Exp. Neurol.
(1994) - et al.
Human immunodeficiency virus-1 Tat protein interacts with distinct proteasomal alpha and beta subunits
FEBS Lett.
(2003) - et al.
Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair
Cell
(2009) - et al.
Tau binds both subunits of calcineurin, And binding is impaired by calmodulin
Biochim. Biophys. Acta
(2008) - et al.
Shared principles in NF-kappaB signaling
Cell
(2008) - et al.
Calcineurin
Adv. Enzymol. Relat. Areas Mol. Biol.
(1988) - et al.
Calcineurin, a calcium- and calmodulin-binding protein of the nervous system
Proc. Natl Acad. Sci. USA
(1979) - et al.
Calcineurin: form and function
Physiol. Rev.
(2000) - et al.
The T-cell transcription factor NFATp is a substrate for calcineurin and interacts with Fos and Jun
Nature
(1993)
Direct interaction and reciprocal regulation between ASK1 and calcineurin–NFAT control cardiomyocyte death and growth
Mol. Cell. Biol.
Et al., Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins
Nat. Immunol.
Proteasomes and other self-compartmentalizing proteases in prokaryotes
Trends Microbiol.
The 26S proteasome: a molecular machine designed for controlled proteolysis
Annu. Rev. Biochem.
Modification of Cul1 regulates its association with proteasomal subunits
Cell Div.
Polo-like kinase interacts with proteasomes and regulates their activity
Cell Growth Differ.
Cited by (22)
Proteomic and metabolomic responses in D-shape larval mussels Mytilus galloprovincialis exposed to cadmium and arsenic
2016, Fish and Shellfish ImmunologyCitation Excerpt :Endoplasmic reticulum protein ERp29 is a ubiquitously expressed endoplasmic reticulum stress-inducible protein that executes protective action by binding to denatured or aggregated cellular proteins thereby facilitating their refolding [25]. Proteasomes play a critical role in ubiquitin-mediated protein degradation through the ubiquitin–proteasome pathway and are involved in cell proliferation, apoptosis and stress responses [26]. He et al. found that the transcription of proteasome in hemocytes was up-regulated in Penaeus japonicus after microbial challenge [27].
Proteomic and metabolomic analysis on the toxicological effects of Benzo[a]pyrene in pearl oyster Pinctada martensii
2016, Aquatic ToxicologyCitation Excerpt :Two significantly changed proteins, cathepsin L and proteasome are involved in protein degradation and apoptosis. These two proteins play a key role in ubiquitin-mediated protein degradation through the ubiquitin-proteasome pathway, and are involved in cell proliferation, stress responses and apoptosis (Aranishi et al., 1997; Zhang et al., 2011a, 2011b; Zhang and Wei, 2011). In the present study, cathepsin L was down-regulated in the low concentration (1 μg/L) of BaP-treated group, inversely it was up-regulated in the high concentration (10 μg/L) of BaP-treated group.
Proteomic and metabolomic responses in hepatopancreas of Mytilus galloprovincialis challenged by Micrococcus luteus and Vibrio anguillarum
2013, Journal of ProteomicsCitation Excerpt :In M. luteus-challenged mussel samples, the responsive proteins (Table 3) related to metabolism were involved in energy and primary metabolisms. Proteasomes play a critical role in ubiquitin-mediated protein degradation through the ubiquitin–proteasome pathway and are involved in cell proliferation, apoptosis and stress responses [41]. He et al. found that the transcription of proteasome in hemocytes was up-regulated in Penaeus japonicus after microbial challenge [42].
Proteomic and metabolomic analysis reveal gender-specific responses of mussel Mytilus galloprovincialis to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47)
2013, Aquatic ToxicologyCitation Excerpt :Septins are cytoskeletal proteins assembling as intracellular filamentous scaffolds involved in diverse cellular biological processes including cell apoptosis (Sui et al., 2003). Proteasomes play a critical role in protein degradation and are involved in cell proliferation, apoptosis and stress responses (Zhang and Wei, 2011). These proteins including TCTP, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation, septin-6, VDAC 2-like protein and proteasome subunit alpha type-2-like were significantly altered in male mussel gills exposed to low concentration (1 μg/L) of BDE 47, which implies cell apoptosis induced by BDE 47.
PEGylated Phthalocyanine-Functionalized Graphene Oxide with Ultrahigh-Efficient Photothermal Performance for Triple-Mode Antibacterial Therapy
2021, ACS Biomaterials Science and Engineering