INTERACTIONS OF MEK &ERK KINASES

University Of Calif-Los Alamos Nat Lab, Los Alamos NM

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Abstract

We are attempting to determine the nature of the signals transmitted to the nucleus that result in cell division. Reversible protein phosphorylation appears to be important in regulating some of these early biochemical events. Therefore, the potentially relevant protein kinases, phosphoprotein phosphatases, and their substrates are purified and characterized. This project seeks to determine the nature of the molecular interactions between Erk (extracellular receptor kinase) and Mek (MAPK/Erk kinase) in this signaling pathway, especially the effect of phosphorylation on kinase activation. We have expressed and purified these proteins as native and GST- and His-tag fusion proteins. In addition we have constructed kinase-inactive and -constitutively active mutants, as well as phosphorylation site mutants. We will use these reagents with a novel flow cytometric technique to measure molecular interactions. GST-Erk will be attached to a glutathione labeled bea d and the binding of fluorescent Mek will be measured. This approach will give us information on binding affinity and dynamics not available from conventional methods. .