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- [PROTOCOL] Determination of protein phosphorylation by polyacrylamide gel electrophoresis
- Chang-Ro Lee , Young-Ha Park , Huitae Min , Yeon-Ran Kim , Yeong-Jae Seok
- J. Microbiol. 2019;57(2):93-100. Published online January 31, 2019
- DOI: https://doi.org/10.1007/s12275-019-9021-y
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Abstract
- Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.
- Stage-specific change and regulation of endogenous protein phosphorylation in allomyces macrogynus
- Park, Young Shik , Oh, Keun Hee , Lee, Soo Woong , Seong, Chang Soo , Park, I Ha , Yim, Jeong Bin
- J. Microbiol. 1996;34(4):374-378.
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Abstract
- In the aquatic fungus Allomyces macrogynus the effects of Ca^2+ and cAMP on the intracellular signal transduction of zeoospore germination were studied using in vitro protein phosphorylation assay system. An endogenuously phosphorylated protein (p50) having molecular weight of 50 kDa on SDS-PAGE was found in soluble fractions of both zeoospore and mycelium. In zoospore extract, the endogenous phophorylation of p50 was weak without any effectors, but was enhanced by Ca^2+ and even more by cAMP. Phosphorylation of the same protein in mycelial extract was high only in the absence of cAMP. Irrespective of the presence of Ca^2+ and cAMP, its phosphorylation was antagonistically suppressed in assay of combined zoospore and mycelial extracts. These results suggest that p50 is interconvertible in phosphorylation/dephosphorylation as a novel protein involved in germination of A. macrogynus. The antagonistic effect of cAMP to the phosphorylation of p50s from different developmental stages may be important in the regulation of cellular differentiation.
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