Recombinant Human CKB protein, His-tagged

• Cat.No.: CKB-495H
• Product Overview: Recombinant Human CKB protein(P12277)(Pro2-Lys381) was expressed in Insect Cells, fused with a polyhistide tag at the N-terminus.

Specification

• Species: Human
• Source: Insect Cells
• Tag: His
• Protein Length: 2-281 a.a.
• Tag: N-His
• Form: Supplied as sterile 50mM Tris, 100mM NaCl, 10% glycerol, pH 8.0.
• Molecular Mass: The recombinant human CKB consists of 398 amino acids and has a calculated molecular mass of 44.8 kDa. The recombinant protein migrates as an approximately 45 kDa band in SDS-PAGE under reducing conditions.
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method
• Purity: > 90 % as determined by SDS-PAGE
• Storage: Samples are stable for up to twelve months from date of receipt at -20°C to -80°C. Store it under sterile conditions at -20°C to -80°C. It is recommended that the protein be aliquoted for optimal storage. Avoid repeated freeze-thaw cycles.
• Reconstitution: It is recommended that sterile water be added to the vial to prepare a stock solution of 0.2 ug/ul. Centrifuge the vial at 4°C before opening to recover the entire contents.
• Publications:
  CKB inhibits epithelial-mesenchymal transition and prostate cancer progression by sequestering and inhibiting AKT activation (2021)

Gene Information

• Gene Name: CKB creatine kinase, brain [ Homo sapiens ]
• Official Symbol: CKB
• Synonyms: CKB; creatine kinase, brain; CKBB; creatine kinase B-type; creatine kinase-B; creatine kinase B chain; B-CK
• Gene ID: 1152
• mRNA Refseq: NM_001823
• Protein Refseq: NP_001814
• MIM: 123280
• UniProt ID: P12277

Citation

CKB inhibits epithelial-mesenchymal transition and prostate cancer progression by sequestering and inhibiting AKT activation

Journal: Neoplasia (New York, N.Y.)    PubMed ID: 34706306    Data: 2021/10/24

Authors: Zheng Wang, Mohit Hulsurkar, Wenliang Li

Article Snippet:Protein samples together with anti-AKT antibody (#2920, Cell Signaling Technology), anti-Rictor antibody (#051471, Millipore sigma) or mouse IgG isotype control antibody (#5415, Cell Signaling Technology) were incubated at 4°C overnight.Protein samples together with anti-AKT antibody (#2920, Cell Signaling Technology), anti-Rictor antibody (#051471, Millipore sigma) or mouse IgG isotype control antibody (#5415, Cell Signaling Technology) were incubated at 4°C overnight.. To detect direct interaction of AKT and CKB in vitro, 2ug purified his-AKT1 (#PR3878D, Invitrogen) and his-CKB (#495H, Creative BioMart) were incubated at 4°C for 2h.. Equal amount of anti-CKB antibody (#ab108388, Abcam) and rabbit IgG isotype control antibody (#3900, Cell Signaling Technology) were added for binding at 4°C overnight.Equal amount of anti-CKB antibody (#ab108388, Abcam) and rabbit IgG isotype control antibody (#3900, Cell Signaling Technology) were added for binding at 4°C overnight.

CKB silencing induces AKT-S473 phosphorylation, consistent with their correlation in patient samples. (A) Whole cell lysate from PC3 cells expressing either shScram or shCKB-1 was tested on a human phospho-kinase array (R&D Systems). Red circles show duplicate spots with signals from p-S473-AKT antibody. (B) Immunoblotting for p-S473-AKT, CKB and Beta Actin in DU145 cells expressing shScram or shCKB-1 (left), and PC3 cells carrying empty vector control (EV, Ctrl) or CKB cDNA (right). (C) Immunoblotting for E-Cadherin, Vimentin, Slug, Twist, p-S473-AKT, AKT and beta-Actin in PC3 parental (WT) and CKB knockout cells. (D) Immunohistochemistry analysis of p-S473-AKT in PC3-shScramble and PC3-shCKB xenografts. (E) p-S473-AKT levels in TCGA primary prostate tumors, as analyzed by RPPA (accessed through cBioportal), comparing samples with low CKB mRNA expression (Z-score <-0.5) with all other samples. The p-S473-AKT plot and P value ( P < 0.01) were obtained from cBioPortal (color version of figure is available online).

AKT inhibitor and CKB overexpression reverse EMT induced by CKB suppression. (A) RT-qPCR result of CKB expression in the indicated 4 cell lines treated with DMSO or 15uM AKT inhibitor MK-2206 for 24 h. Expression pattern for epithelial marker Occludin from these experiments is in Figure S4B. (B) Parental PC3 cells (wild type, WT), PC3 with CKB CRISPR-Cas9 knockout (CKB KO), or PC3 with CKB knockdown (shCKB-1) were treated with DMSO or 3uM of AKT inhibitor MK-2206 for 48h. Immunoblots for indicated proteins were shown. (C) qPCR analysis for indicated genes normalized to beta-Actin in PC3 parental cells (WT), CKB knockout cells (CKB KO), and CKB-knockout cells re-expressing CKB cDNA (CKB KO + CKB). * P < 0.05, ** P < 0.01 comparing CKB KO to CKB KO + CKB. (D) Cell morphology were monitored by phase-contrast microscopy in PC3 WT, CKB KO and CKB KO + CKB cells. (E) Boyden chamber migration assay for PC3 WT cells, PC3 CKB KO cells and PC3 CKB KO + CKB cells. (F) Quantitative results of the migrations in E, based on triplicates. *** P < 0.001, **** P < 1 × 10 ?4 , ***** P < 1 × 10 ?5 . (G) Alamar blue cell proliferation assay for PC3 WT, CKB KO and CKB KO + CKB cells cultured in media with reduced FBS (1.25%) at day 1, 3, 5 and 7 (quadruplicates). ** P < 0.01, *** P < 0.001 comparing CKB KO cells, CKB KO + CKB cDNA cells with WT cells at corresponding time points. ^^ P < 0.01 comparing CKB KO with CKB KO + CKB cells at day 7. These experiments have been repeated at least twice, which has yielded same conclusions. Results from a representative experiment are shown.

CKB interacts with AKT and inhibits AKT activation. (A) CKB and AKT proteins could reciprocally co-immunoprecipitate (co-IP) each other from LNCaP cells. (B) AKT protein was immunoprecipitated by CKB antibody in a mixture of recombinant His-tagged AKT and His-tagged CKB proteins. (C) PC3-GFP and PC3-CKB cells were treated with or without 100ng/ml EGF for 5 min. Endogenous AKT was immunoprecipated from these 2 cell lines using AKT Ab, followed by immunoblotting for Rictor, mTOR and AKT (top). Conversely, endogenous Rictor was immunoprecipated using Rictor Ab, then immunoblot for AKT, mTOR and Rictor (middle). Immunoblotting of the input whole cell lysates was shown in bottom. Fold changes of Rictor and AKT protein levels in the IP samples are plotted, relative to the sample without EGF and without CKB overexpression. The quantification is based on measurements from 2 independent experiments, using ImageJ software.

Case Study

Case 1: Wang Z, et al. Neoplasia. 2021

EMT aids cancer spread and resistance, with AKT being crucial. Typically, there's a lack of inhibitors, but brain-type creatine kinase (CKB) has emerged as a suppressor. While normally present, CKB decreases in some cancers, leading to worse outcomes. When CKB is boosted, EMT and cancer movement lessen; when reduced, cancer activity spikes. CKB blocks AKT activation by binding to part of it, and even just an 84aa fragment of CKB can slow AKT, EMT, and cancer cell growth.

Fig1. Immunoblotting of whole cell lysates of PC3 cells stably expressing scramble control shRNA or CKB shRNA.

Fig2. Immunoblotting for p-S473-AKT, CKB and Beta Actin in DU145 cells.

Case 2: Rahbani JF, et al. Nature. 2021

Obesity brings higher risks for diseases like diabetes and heart issues. But adipocytes, through heat production, can help tackle obesity. In this process, creatine helps mitochondria make heat, but researchers didn't know which proteins handled this. It turns out creatine kinase B (CKB) is key, moving to mitochondria to do its job. When CKB kicks in, it’s triggered by heat in both mice and humans. If mice lack CKB in fat cells, they can't produce as much heat, gain weight easier, and struggle with glucose control. So, CKB is essential in this energy-burning cycle.

Fig1. Western blot of BAT from wild-type (C57BL6/N, 6-8 weeks old) male mice.

Fig2. TargetP probability plotted along the CKB amino acid sequence.

Application

Recombinant CKB protein is quite promising for therapeutic use, especially in managing obesity. Since CKB is crucial for burning energy through thermogenesis, it could help boost the body's calorie-burning ability, making it a valuable tool in tackling obesity and related metabolic issues. Apart from weight control, CKB is also being looked at for muscle support. It's vital for energy use in cells, possibly aiding recovery from muscle injuries or boosting athletic performance. This could make it useful in muscle rehab and improving endurance. CKB's role in heart health is another exciting area. It plays a part in energy use, so recombinant CKB might help in conditions where heart energy is low. This approach could open up new ways to treat heart diseases. Overall, recombinant CKB protein shows great potential for improving metabolic health, muscle performance, and heart function.

Fig1. Regulation of brain-type creatine kinase (CKB) in Huntington’s disease (HD). (Tz-Chuen Ju, 2012)