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1.
J Proteomics ; 255: 104485, 2022 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-35065289

RESUMO

Protein kinases regulate almost all biological processes including cell proliferation, differentiation, apoptosis, and gene expression. Dysregulation of protein phosphorylation caused by abnormal activity and expression of protein kinases results in the onset of various diseases such as cancer and metabolic syndromes. The activities of a large number of protein kinases are regulated by phosphorylation. Therefore, analysis of the phosphorylation status of protein kinases is important for elucidation of biological phenomena and the pathogenesis of diseases. To investigate protein phosphorylation, phosphate-binding tag molecule "Phos-tag" was developed. In addition, various techniques and tools using Phos-tag such as Phos-tag SDS-PAGE, have been developed for analysis and profiling of protein phosphorylation. Here, we describe the methods and analytical techniques that use Phos-tag for investigation of protein kinase phosphorylation and the applications of phosphorylation analysis. SIGNIFICANCE: Protein kinases play pivotal roles in regulating many biological processes and pathogenesis of diseases. Determination of phosphorylation status of protein kinases can provide the essential information for their activation. This review provides analytical techniques for analysis of phosphorylation status of protein kinases by Phos-tag SDS-PAGE. We believe that this review would help readers to study in kinomics research.


Assuntos
Fosfoproteínas , Proteínas Quinases , Eletroforese em Gel de Poliacrilamida , Fosfoproteínas/análise , Fosforilação , Proteínas Quinases/metabolismo , Piridinas
2.
J Appl Glycosci (1999) ; 67(3): 95-101, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-34354535

RESUMO

D-Allose (D-All), a C-3 epimer of D-glucose (D-Glc), is a naturally rare monosaccharide, which shows anti-proliferative activity against several human cancer cell lines. Unlike conventional anticancer drugs, D-All targets glucose metabolism and is non-toxic to normal cells. Therefore, it has attracted attention as a unique "seed" compound for anticancer agents. However, the anti-proliferative activities of the other rare aldohexoses have not been examined yet. In this study, we evaluated the anti-proliferative activity of rare aldohexoses against human leukemia MOLT-4F and human prostate cancer DU-145 cell lines. We found that D-All and D-idose (D-Ido) at 5 mM inhibited cell proliferation of MOLT-4F cells by 46 % and 60 %, respectively. On the other hand, the rare aldohexoses at 5 mM did not show specific anti-proliferative activity against DU-145 cells. To explore the structure-activity relationship of D-Ido, we evaluated the anti-proliferative activity of D-sorbose (D-Sor), 6-deoxy-D-Ido, and L-xylose (L-Xyl) against MOLT-4F cells and found that D-Sor, 6-deoxy-D-Ido, and L-Xyl showed no inhibitory activity at 5 mM, suggesting that the aldose structure and the C-6 hydroxy group of D-Ido are important for its activity. Cellular glucose uptake assay and western blotting analysis of thioredoxin-interacting protein (TXNIP) expression suggested that the anti-proliferative activity of D-Ido is induced by inhibition of glucose uptake via TXNIP-independent pathway.

3.
Biochim Biophys Acta Proteins Proteom ; 1867(1): 71-75, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29753089

RESUMO

Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Anticorpos Monoclonais , Eletroforese em Gel de Poliacrilamida/métodos , Células HL-60 , Humanos , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteômica/métodos
4.
Anal Biochem ; 513: 47-53, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27565380

RESUMO

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.


Assuntos
Fosfoproteínas Fosfatases/química , Proteínas Quinases/química , Animais , Humanos , Camundongos , Fosforilação , Especificidade por Substrato
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