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.
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