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Molecular and Cellular Biology, March 2006, p. 2065-2079, Vol. 26, No. 6
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.6.2065-2079.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

PB1 Domain-Dependent Signaling Complex Is Required for Extracellular Signal-Regulated Kinase 5 Activation

Kazuhiro Nakamura, Mark T. Uhlik, Nancy L. Johnson, Klaus M. Hahn, and Gary L. Johnson*

Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7365

Received 28 October 2005/ Returned for modification 22 November 2005/ Accepted 22 December 2005

MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.

* Corresponding author. Mailing address: Department of Pharmacology, CB#7365, 1108 Mary Ellen Jones Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. Phone: (919) 843-3107. Fax: (919) 966-5640. E-mail: gary_Johnson{at}med.unc.edu.

Molecular and Cellular Biology, March 2006, p. 2065-2079, Vol. 26, No. 6
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.6.2065-2079.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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