Interactions between RaF proto-oncogenes and CDC25 phosphatases, and uses related thereto
First Claim
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1. A method for identifying a compound which is an inhibitor of ras-mediated activation of a CDC25 phosphatase, comprising the steps of:
- a) generating a reconstituted protein combination including;
1) a test agent to be assessed;
2) a purified protein preparation of a mammalian CDC25 phosphatase,3) a purified protein preparation of a kinase from a ras-mediated signal transduction cascade, which kinase phosphorylates the CDC25 phosphatase; and
4) a substrate of the CDC25 phosphatase;
b) maintaining the combination under conditions appropriate for the kinase to phosphorylate the CDC25 phosphatase and for an activated CDC25 phosphatase to convert the substrate to product; and
c) measuring the conversion of the substrate to product,wherein a decrease in the conversion of substrate to product in the combination, relative to a control comprising the CDC25 phosphatase, the kinase, and the substrate and lacking the test agent, indicates that the test compound is an inhibitor of ras-mediated activation of the CDC25 phosphatase.
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Abstract
The present invention derives from the discovery that CDC25 phosphatases and Raf proteins are able to physically interact to form protein-protein complexes, with the Raf protein mediating the activation of CDC25 phosphatases. The present invention provides both cell-free and cellular assays for detecting agents which modulate the ras-dependent activation of CDC25, as for example, by affecting the binding of a CDC25 protein with Raf, or Raf-associated complexes. Also disclosed is a method for transforming/immortalizing cells, particularly primary cell cultures.
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16 Claims
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1. A method for identifying a compound which is an inhibitor of ras-mediated activation of a CDC25 phosphatase, comprising the steps of:
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a) generating a reconstituted protein combination including; 1) a test agent to be assessed; 2) a purified protein preparation of a mammalian CDC25 phosphatase, 3) a purified protein preparation of a kinase from a ras-mediated signal transduction cascade, which kinase phosphorylates the CDC25 phosphatase; and 4) a substrate of the CDC25 phosphatase; b) maintaining the combination under conditions appropriate for the kinase to phosphorylate the CDC25 phosphatase and for an activated CDC25 phosphatase to convert the substrate to product; and c) measuring the conversion of the substrate to product, wherein a decrease in the conversion of substrate to product in the combination, relative to a control comprising the CDC25 phosphatase, the kinase, and the substrate and lacking the test agent, indicates that the test compound is an inhibitor of ras-mediated activation of the CDC25 phosphatase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification