Extracellular Signal-Regulated Kinase 2 (ERK2) Phosphorylation Sites and Docking Domain on the Nuclear Pore Complex Protein Tpr Cooperatively Regulate ERK2-Tpr Interaction

Abstract

Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

ACKNOWLEDGMENTS

This work is supported by intramural and extramural funding provided by the Department of Biotechnology, India, to V.K.N., grants R01 CA105402, P01 CA040042, and P01 CA076465 to M.J.W. from the National Institute of Health and the Academy of Sciences of the Czech Republic Institutional Research Concept AV0Z50200510, and grant IAA500200716 to T.V.

D.T. is a Senior Research Fellow of the Council of Scientific and Industrial Research.

We thank members of the Parsons-Weber-Parsons group for helpful discussions; Scott Eblen for the FLAG-ERK2-TAYF and FLAG-ERK2-K52R constructs; Larry Gerace, Melanie Cobb, Dennis J. Templeton, and Roger Davis for materials used in this study; John Coyle, Marie-Louise Hammarskjold, and David Rekosh for assistance in testing the effects of Tpr phosphorylation on nuclear transport; and Swati Saha for critical reading of the manuscript.