ABSTRACT
The CacyBP/SIP protein is expressed at a particularly high level in brain, spleen, and various tumors. In this work, we have studied transcriptional regulation of the CacyBP/SIP gene and the influence of increased CacyBP/SIP level on gene expression in colorectal cancer HCT116 cells. We have shown that E2F1, EGR1, and CREB transcription factors bind to the CacyBP/SIP gene promoter and stimulate transcription of CacyBP/SIP gene. The role of CREB was further confirmed by the observation that forskolin, a strong activator of CREB phosphorylation/activity, increased CacyBP/SIP gene promoter activity. Moreover, we have shown that CREB dominant negative mutants, CREB133 and KCREB, inhibits CacyBP/SIP promoter activity. To check the biological significance of increased CacyBP/SIP expression/level we have applied RNA microarray analysis and have found that upregulation of CacyBP/SIP entails changes in mRNA level of many genes involved, among others, in immune processes. © 2017 IUBMB Life, 70(1):50-59, 2018.
Subject(s)
Calcium-Binding Proteins/genetics , Cyclic AMP Response Element-Binding Protein/genetics , E2F1 Transcription Factor/genetics , Early Growth Response Protein 1/genetics , Gene Expression Regulation, Neoplastic , Transcriptional Activation , Binding Sites , Calcium-Binding Proteins/metabolism , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Colforsin/pharmacology , Cyclic AMP Response Element-Binding Protein/metabolism , E2F1 Transcription Factor/metabolism , Early Growth Response Protein 1/metabolism , Gene Expression Profiling , Genes, Reporter , HCT116 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Oligonucleotide Array Sequence Analysis , Phosphorylation , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal TransductionABSTRACT
In this work we have shown that NFAT1 transcription factor is involved in the regulation of CacyBP/SIP expression. We have demonstrated, by applying Western blot, RT-PCR and luciferase assay that the level of CacyBP/SIP increases upon NFAT1 overexpression. Moreover, inhibition or stimulation of NFAT transcriptional activity exerts a corresponding effect on the expression of CacyBP/SIP gene. Furthermore, EMSA and chromatin immunoprecipitation (ChIP) assay have shown that NFAT1 binds to its specific binding sites within the CacyBP/SIP gene. In conclusion, our data have shown for the first time the regulation of CacyBP/SIP gene expression by NFAT1. Since NFAT transcription factors are involved in processes related to immune response, these results indicate potential involvement of CacyBP/SIP in the immune system.
Subject(s)
Calcium-Binding Proteins/genetics , NFATC Transcription Factors/genetics , Binding Sites , Calcium-Binding Proteins/metabolism , Cell Line, Tumor , Gene Expression Regulation , Humans , NFATC Transcription Factors/antagonists & inhibitorsABSTRACT
The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.
Subject(s)
Cell Cycle Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Nuclear Proteins/metabolism , S100 Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , Humans , Protein Binding , S100 Calcium Binding Protein A6 , Signal TransductionABSTRACT
In this work, we have found that casein kinase II (CKII) phosphorylates the CacyBP/SIP protein under in vitro conditions and have mapped the phosphorylation site to threonine 184. Moreover, we present evidence that S100A6, a CacyBP/SIP interacting protein, inhibits this phosphorylation in the presence of Ca(2+). CacyBP/SIP phosphorylation by CKII was also observed in neuroblastoma NB2a cells. Interestingly, we have found that the effect of DRB, a CKII inhibitor, on CacyBP/SIP phosphorylation state is similar to that of S100A6 overexpression. Phosphorylation at threonine 184 seems to have an effect on CacyBP/SIP phosphatase activity since the T184E phosphorylation mimic mutant overexpressed in NB2a cells has lower phosphatase activity toward p-ERK1/2 when compared to the non-phosphorylable T184A mutant or to the wild-type protein. In conclusion, our data suggest that S100A6 and Ca(2+), through inhibiting CacyBP/SIP phosphorylation on threonine 184, are important regulators of CacyBP/SIP phosphatase activity and of ERK1/2-Elk-1 signaling pathway.
Subject(s)
Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , S100 Proteins/metabolism , ets-Domain Protein Elk-1/metabolism , Animals , Calcium-Binding Proteins/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Dichlororibofuranosylbenzimidazole/pharmacology , Mice , Neuroblastoma/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , S100 Calcium Binding Protein A6 , S100 Proteins/geneticsABSTRACT
Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates ß-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates ß-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled ß-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for ß-alanine, and a KM of ~ 5 µm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that ß-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a ß-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.