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Molecular and Cellular Biology, May 2003, p. 3067-3078, Vol. 23, No. 9
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.9.3067-3078.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane Region

Perry M. Chan,1,{dagger} Subburaj Ilangumaran,1 Jose La Rose,1 Avijit Chakrabartty,2 and Robert Rottapel1,2,3,4*

Division of Experimental Therapeutics, Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario M5G 2M9,1 Department of Medical Biophysics,2 Departments of Immunology and Medicine, University of Toronto, Toronto, Ontario M5S 1A8,3 St. Michael's Hospital, Toronto, Ontario M5B 1W8, Canada4

Received 24 June 2002/ Returned for modification 8 August 2002/ Accepted 27 January 2003

Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.

* Corresponding author. Mailing address: Division of Experimental Therapeutics, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Phone: (416) 946-2233. Fax: (416) 946-2984. E-mail: rottapel{at}uhnres.utoronto.ca.

{dagger} Present address: Glaxo-IMCB Group, Institute of Molecular and Cell Biology, Singapore 117609, Singapore.

Molecular and Cellular Biology, May 2003, p. 3067-3078, Vol. 23, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.9.3067-3078.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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