Casein kinase 2 interacts with and phosphorylates ataxin-3

Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.

Inflammation-mediated age-dependent effects of casein kinase 2-interacting protein-1 on osteogenesis in mesenchymal stem cells

Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain unclear.This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.Methods:Bone marrow MSCs of CKIP-1 wild type(WT)and knockout(KO)mice were cultivated in vitro.Cell phenotype was analyzed by flow cytometry,colony formation was detected to study the proliferative ability.Osteogenic and adipogenic induction were performed.The osteogenic ability was explored by alizarin red staining,alkaline phosphatase(ALP)staining and ALP activity detection.Quantitative real-time polymerase chain reaction(qRT-PCR)was carried out to determine the mRNA expression levels of osteoblast marker genes.The adipogenic ability was detected by oil red O staining.Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume(BV/TV),bone surface area fraction/trabecular BV,trabecular number(Tb.N),and trabecular spacing(Tb.sp).Interleukin(IL)-1b was injected on WT mice of 2 months old and 18 months old,respectively.Difference in CKIP-1 expression was detected by RT-PCR and western blot.The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.Results:ALP assays,alizarin red staining,and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis.Micro-computed tomography detection showed that among 18-month-old mice,CKIP-1 KO mice presented significantly higher bone mass compared withWTmice(P=0.02).No significant difference was observed in 2-month-old mice.In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice(4.3-fold increase at themRNA level,P=0.04).Finally,the expression levels of CKIP-1 in bone marrow(3.2-fold increase at themRNA level,P=0.03)and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1b.Conclusions:CKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects.Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.

Genetically engineered M2-like macrophage-derived exosomes for P.gingivalis-suppressed cementum regeneration:From mechanism to therapy

Cementum,a thin layer of mineralized tissue covering tooth root surface,is recognized as the golden standard in periodontal regeneration.However,current efforts mainly focus on alveolar bone regeneration rather than cementum regeneration,and rarely take Porphyromonas gingivalis(Pg),the keystone pathogen responsible for periodontal tissue destruction,into consideration.Though M2 macrophage-derived exosomes(M2-EXO)show promise in tissue regeneration,the exosome-producing M2 macrophages are induced by exogenous cytokines with transitory and unstable effects,restricting the regeneration potential of M2-EXO.Here,exosomes derived from genetically engineered M2-like macrophages are constructed by silencing of casein kinase 2 interacting protein-1(Ckip-1),a versatile player involved in various biological processes.Ckip-1 silencing is proved to be an effective gene regulation strategy to obtain permanent M2-like macrophages with mineralization-promoting effect.Further,exosomes derived from Ckip-1-silenced macrophages(sh-Ckip-1-EXO)rescue Pg-suppressed cementoblast mineralization and cementogenesis.Mechanismly,sh-Ckip-1-EXO delivers Let-7f-5p targeting and silencing Ckip-1,a negative regulator also for cementum formation and cementoblast mineralization.More deeply,downregulation of Ckip-1 in cementoblasts by exosomal Let-7f-5p activates PGC-1α-dependent mitochondrial biogenesis.In all,this study provides a new strategy of genetically engineered M2-like macrophage-derived exosomes for cementum regeneration under Pg-dominated inflammation.

Effects of siRNA specific to the protein kinase CK2α on apoptosis of laryngeal carcinoma cells

Background The relationship between apoptosis and tumors is a major focus in cancer research. RNA interference （RNAi） technology has emerged as a very potent tool to generate cellular knockdown phenotypes of a desired gene. The aim of this study was to explore the effect of siRNA specific to the protein casein kinase 2α （CK2α） on apoptosis of laryngeal carcinoma cells and to explore possible mechanisms.Methods An siRNA expression plasmid specific to CK2α, psiRNA-hH1neo-CK2α, and a non-specific siRNA expression plasmid, psiRNA-hH1neo-cont, were constructed and transfected into Hep-2 cells by a lipofectamine method. The mRNA and protein levels of CK2α in transfected cells were determined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting analysis. Apoptosis was measured by Annexin V-fluorescein isothiocyanate （FITC）/ propidium iodide （PI） double-staining methods. The morphological changes to Hep-2 cells were observed under transmission electron microscopy （TEM）. The levels of Bcl-2 and Bax proteins were measured by Western blotting analysis.Results Levels of CK2α mRNA and protein were significantly decreased in the psiRNA-hH1neo-CK2α group compared to the other groups （P 〈0.05）. The apoptotic rate of the psiRNA-hH1neo-CK2α transfected group was significantly higher compared to that in the untransfected group and the siRNA-hH1neo-cont transfected group （25.66%±0.83%, 3.66%±0.43%, and 5.18%±0.22%） （P 〈0.05）. Compared with the untransfected group and the siRNA-hH1neo-cont transfected group, the psiRNA-hH1neo-CK2α transfected group presented with classical ultrastructural features of apoptosis, such as karyopyknosis, chromatic agglutination adjacent to the nuclear membrane, and apoptotic bodies. Compared with the other two groups, the level of Bcl-2 protein in the psiRNA-hH1neo-CK2α transfected group was decreased （0.20±0.09 vs. 0.72±0.16, 0.56±0.11, P 〈0.01）, while the Bax protein level was increased （0.81±0.17 vs. 0.26±0.12, 0.33±0.17, P 〈0.01） and the ratio of Bcl-2 to Bax was decreased （0.25±0.05 vs. 2.76±0.21, 1.70±0.22, P 〈0.01）.Conclusions The siRNA expression plasmid specific to CK2α could suppress CK2α expression and induce the apoptosis of laryngeal carcinoma cells. This is possibly by decreasing the Bcl-2/Bax ratio. CK2α may provide a potential therapeutic target against human laryngeal carcinoma.

Identifcation of the Phosphorylated Residues in TveIF5A by Mass Spectrometry

The initiation factor elF5A in Trichomonas vaginalis （TvelF5A） is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TvelF5A have been reported. The most acidic isoform （pI 5.2） corresponds to the precursor TvelF5A, whereas the mature TvelF5A appears to be the most basic isoform （pI 5.5）. In addition, the intermediary isoform （pI 5.3） is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TvelF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TvelF5A contains four phosphorylated residues （$3, T55, T78 and T82）. Phosphorylation at $3 and T82 is also identified in the intermediary TvelF5A, while no phosphorylated residues are found in the precursor TvelF5A. It has been demonstrated that elF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 （CK2）. Interestingly, a gene encoding a protein highly similar to CK2 （TvCK2） is found in T. vaginalis, which might be involved in the phosphorylation of TvelF5A in T. vaginalis.