Laboratory of Molecular Biophysics Laboratory Journal 2000 Prof. L. N. Johnson

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Louise N. Johnson

Protein kinases, protein phosphatases and molecular recognition.

Introduction

Our major interests are in understanding the structural basis of cellular control processes by phosphorylation events and, more recently, in the mechanisms which target proteins for ubiquitination and destruction. The eukaryotic protein kinase domain is the first, second and third most abundant domain in the genome sequences of yeast, worm and fly, respectively, indicating the importance of phospho-signaling in eukaryotes. More than 400 eukaryotic kinases have been identified and it is expected that the human genome will contain about 1000 kinases. The 21 eukaryotic protein kinase structures solved to date show that kinases comprise a common fold, in which the constellation of atoms and lobe orientation in the active conformation have considerable structural identity but that each inactive kinase is inactive in its own way. Our work has focused on the fascinating structural problems of how kinases respond to cellular signals through conformational changes and binding of regulatory domains or subunits and the manner in which different kinases are targeted to their specific substrates using both local sequence recognition motifs and more remote targeting sites.

This year our work divides into studies on the regulatory proteins of the cell cycle, the mechanism of catalysis of protein kinases, the control of phosphorylase kinase by calcium/calmodulin and the recognition properties of protein phosphatases. With the cell cycle proteins, we have solved the structure of the complex between the cell cycle regulatory kinase CDK2 and the regulatory phosphatase (KAP) to reveal details of phospho-protein recognition and conformational response; we have identified the sites of regulatory phosphorylation in the cyclin dependent activating kinase (CDK7/H) when expressed in baculoviral infected insect cells; we have developed a strategy for further investigation of the relationship between the substrate recognition catalytic site and the substrate recruitment site for the active phospho-CDK2/cyclin A complex, and we have begun studies on a new protein, DRP1, that interacts with polyubiquitinated cyclin A and prevents destruction by the proteasome. In continuation of work on catalytic mechanism we have studied a potential transition-state complex structure with pCDK2/cyclin A and established a template that for the catalytic site residues of active kinases.

With phosphorylase kinase we have developed a new expression strategy to enable us to produce soluble complex of the phosphorylase kinase with calmodulin in order to elucidate the structural basis of control by calcium. Phosphorylase kinase holoenzyme, a large complex of molecular weight 1.6 x 10⁶, is being studied by electron microscopy (C. Vénien). With protein phosphatases, Annette Salmeen working with David Barford has established the structural basis for the recognition of the tri-phosphorylated peptide from the insulin receptor tyrosine kinase domain, work that has relevance for the turning off of the insulin signal and for diabetes and studies on the recognition properties of the MAP kinases phosphatase, MKP3, have progressed. In collaboration with Jane Endicott and Martin Noble, we have a continuing interest in structure based drug design with CDK2 leading to new compounds of potential application in the control of cancer. Finally in preliminary work we are establishing a bioinformatics approach to the study of kinase recognition surfaces.

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