Allosteric Activation of ErbB2 by ErbB3 via Tethering of the Kinase Domains

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	FDA/CBER	BIOCHEM-2

Authors

• RS De Silva
• L Wong
• YX Fan
• GR Johnson

Abstract

Activation of the EGF receptor tyrosine kinase arises from formation of an asymmetric dimer in which the C-lobe of one kinase domain (KD) interacts with the N-lobe of the other KD. However, there is no direct evidence that ErbB3 utilizes the same mechanism to activate the related ErbB2 kinase. Here, we have employed a strategy that facilitates the study of the activated ErbB3/ErbB2 kinase heterodimer in solution by generating a fusion containing both KDs. Robust activation was observed when the linked domains simultaneously contained the juxtamembrane region of ErbB2 and a 39 amino acid segment which abuts the C-terminus of the ErbB3 KD. The allosteric activation resulted in an ~7-fold increase in the catalytic rate constant (kcat), and mutations in the ErbB3 helix αH completely reversed this activation. In cells the identical mutations in helix αH, but not those in the N-lobe or that disrupt nucleotide binding, result in full-length ErbB3s that are incapable of mediating heregulin-induced transactivation of ErbB2. These findings provide strong evidence that the C-lobe of the ErbB3 KD directly engages and activates the ErbB2 KD in a heterodimer and this novel approach may be useful in the biochemical characterization of the activation mechanisms of protein kinases.

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