Methods for measuring protein kinase and phosphatase activity

A method for determining the dephosphorylating activity of an enzyme acting on a substrate molecule, said substrate comprising at least one phosphorylated moiety that is capable of being de-phosphorylated by said enzyme to yield a product and wherein said substrate is labelled with a fluorescent dye selected from the acridone and the quinacridone classes of dyes, said method comprising the steps of:

• i) measuring the fluorescence intensity of the fluorescently labelled substrate;
• ii) combining said enzyme with said substrate molecule to yield a product;
• and
• iii) measuring a decrease in fluorescence intensity following the combination of step ii);
• wherein said decrease in fluorescence intensity of the fluorescent label is used to determine the dephosphorylating activity of said enzyme.

A method for determining the phosphorylation status of a substrate in a cellular environment, the substrate comprising at least one moiety that is capable of being phosphorylated or dephosphorylated by a cellular enzyme to yield a product, and wherein said substrate is labelled with a fluorescent dye, said method comprising the steps of:

• i) measuring the fluorescence intensity and the fluorescence lifetime of the fluorescently labelled substrate in a cell-free environment;
• ii) adding the substrate to one or more cells in a fluid medium;
• and
• iii) measuring the fluorescence intensity and the fluorescence lifetime of the fluorescent label following step ii);
• wherein a change in the fluorescent intensity and the fluorescence lifetime is used to indicate the phosphorylation status of said substrate.

A method of simultaneously measuring the kinase activities of two or more different enzymes each enzyme specific for a different substrate, wherein each substrate comprises at least one moiety that is capable of being phosphorylated by an enzyme to yield a product, and wherein each said substrate is labelled with a different one of a set of fluorescent dyes, said method comprising the steps of:

• i) measuring the fluorescence intensity and the fluorescence lifetime of each of the fluorescently labelled substrates;
• ii) combining a mixture of said enzymes with each of said substrate molecules in the presence of a phosphate source;
• and
• iii) measuring an increase in fluorescence intensity and in fluorescence lifetime of each of the fluorescent labels following the combination of step ii);
• wherein said increase in fluorescence intensity and in fluorescence lifetime of each of the fluorescent labels is used to determine the phosphorylating activity of each of said enzymes.