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J. Biol. Chem., Vol. 283, Issue 3, 1588-1596, January 18, 2008

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Mutational Activation of ErbB2 Reveals a New Protein Kinase Autoinhibition Mechanism^*

Ying-Xin Fan¹, Lily Wong, Jinhui Ding, Nikolay A. Spiridonov, Richard C. Johnson^¶, and Gibbes R. Johnson²

From the Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration and Bioinformatics Section, Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda Maryland 20892 and ^¶Department of Neuroscience, Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, Maryland 21205

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the C helix and β4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to 10- and 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the C-β4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the C-β4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the C-β4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of C-β4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

Received for publication, September 28, 2007 , and in revised form, November 13, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1S and 2S.

¹ To whom correspondence may be addressed: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bldg. 29A, Rm. 3B-20, 8800 Rockville Pike, Bethesda, MD 20892. E-mail: ying-xin.fan{at}fda.hhs.gov.

² To whom correspondence may be addressed: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bldg. 29A, Rm. 3B-16, 8800 Rockville Pike, Bethesda, MD 20892. E-mail: gibbes.johnson{at}fda.hhs.gov.

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