Assay for activity of the ActRIIB kinase

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The present invention relates to kinase assays, and specifically to novel kinase assay methods using a novel target peptide for measuring the activity and/or modulation of activity of the ActRIIB kinase protein.

In a first aspect, the present invention provides an assay for determining the kinase activity of ActRIIB, said assay comprising the steps of incubating ActRIIB, labelled phosphodonor, and a target peptide in an suitable medium such that said ActRIIB may phosphorylate said target peptide using said labelled phosphodonor, thereby creating a labelled target peptide, and measuring the amount of label present in said labelled target peptide.

In a second aspect, the present invention provides An assay for discovering modulators of ActRIIB kinase, said assay comprising the steps of incubating in a first reaction ActRIIB kinase, labelled phosphodonor, and a target peptide in an appropriate medium such that said ActRIIB kinase may phosphorylate said target peptide using said labelled phosphodonor, thereby creating a labelled target peptide, and incubating in a second reaction ActRIIB kinase, labelled phosphodonor, target peptide, and a test compound, measuring the amount of label present in said labelled target peptide from said first and second reactions, and comparing the amount of labelled target peptide in said second reaction to the amount of labelled target peptide from said first reaction, wherein any statistically significant difference in the amount of labelled target peptide indicates that said test compound is a modulator of ActRIIB kinase.

In a third aspect, the present invention provides the protein catActRIIB kinase, wherein said protein has the sequence shown in SEQ ID NO:2.

DETAILED DESCRIPTION

Those skilled in the art will fully understand the terms used herein in the description and the appendant claims to describe the present invention. Nonetheless, unless otherwise provided herein, the following terms are as described immediately below.

By “ActRIIB” is meant the protein Activin Receptor IIB, or sometimes the gene encoding the Activin Receptor IIB protein. “ActRIIB kinase” is equivalent to simply “ActRIIB”, though it is typically used when it is desirable to emphasize the kinase activity and/or the kinase catalytic portion of ActRIIB. Unless specified, ActRIIB refers to the protein as found in human, mouse, or any other organism (See, for example, Hilden et al., Blood, 83(8):2163-70 (1994) for human; Attisano et al., Cell, 68:97-108 (1992) for mouse; and Mathews et al., Science, 255:1702-05 (1992) for Xenopus). Additionally, unless specified, ActRIIB refers to all naturally occurring alleles of the protein, as well as man-made (e.g., genetically engineered) variants thereof.

By “catActRIIB” is meant a modified version of the ActRIIB protein designed for use in the present invention. ActRIIB is modified by retaining the catalytic portion and removing hydrophobic portions that are unnecessary for enzymatic activity, thus creating an enzyme with superior solubility characteristics. While unmodified ActRIIB will work in the inventions methods, this modified version is presently preferred. catActRIIB preferably consists of amino acids 167-512 of human ActRIIB (SEQ ID NO:2). These amino acids correspond to the catalytic (cytosolic) domain of the protein, and also correspond to nucleotides 503-1543 of accession #77533 (see Hilden at al., Blood, 83(8):2163-70 (1994)).

By “his-ActRIIB” “his-catActRIIB” is meant the ActRIIB or catActRIIB protein expressed from a construct that has been genetically engineered such that the protein includes a histidine or his6 tag for additional ease of purification.

By “phosphodonor” is meant a compound that is involved in the enzymatic reaction catalyzed by ActRIIB, wherein the phosphodonor loses a phosphoryl group that is transferred to the substrate by ActRIIB. The most common phosphodonors are nucleotide triphosphates and diphosphates. The presently most preferred phosphodonor for use in the present invention is ATP.

By “reduced” is meant a statistically significant decrease (i.e., p<0.1).

By “increased” is meant a statistically significant increase (i.e., p<0.1).

By “modulates” is meant a statistically significant increase or decrease (including a complete elimination).

In its most basic form, a kinase assay is simply a combination of a kinase protein, an appropriate substrate, a phosphate donor, and a means for measuring the amount of reaction that occurs. Of course, within this simplistic plan can lie a multitude of complications. In the case of the present invention, a problem occurred when it was attempted to create an assay for measuring the kinase activity of ActRIIB, or more specifically, a modified version of ActRIIB (referred to herein as catActRIIB, see hereinbelow). A commonly used substrate for kinase assays is Myelin Basic Protein, or MBP. It is generally a good target for the majority of kinases, and is easily and cheaply obtained. However, when it was attempted to use MBP as a substrate for measuring the kinase activity of catActRIIB, the attempt surprisingly failed (see Examples 1 and 2 herein). Thus, it was necessary for another substrate to be used.