The SH2 Domain of BCR-ABL1 Regulates Kinase Autophosphorylation By Controlling Activation Loop Accessibility
Authors: Hantschel Oliver D. , Lamontanara Allan Joaquim , Georgeon Sandrine , Tria Giancarlo , Svergun Dmitri 
Published: October 2019

Chronic myelogenous leukemia (CML) is caused by BCR-ABL1, which is a constitutively active form of the Abelson tyrosine kinase (ABL1). While treatment with the tyrosine kinase inhibitors imatinib, nilotinib, dasatinib, bosutinib or ponatinib that target the ATP binding pocket of BCR-ABL1 leads to durable cytogenetic and molecular remissions in the majority CML patients, primary and secondary drug resistance remains a clinical problem. Targeting additional sites in the BCR-ABL1 kinase outside the highly conserved ATP binding pocket may be an alternative strategy to restrict drug resistance and limit side effects of ATP-competitive drugs with low selectivity. Our recent work has shown that an allosteric intramolecular interaction of the BCR-ABL1 SH2 domain with its kinase domain is critical for leukemogenesis and can be targeted with an engineered high-affinity binding protein.

We have now elucidated the molecular mechanisms responsible for the regulation of BCR-ABL1 kinase activity by its SH2 domain: To this end, we set-up an efficient expression system for the BCR-ABL1 SH2-kinase domain unit in E.coli with excellent yield, purity and activity. Detailed biophysical and biochemical analysis of the purified recombinant proteins in vitro recapitulated SH2-dependent regulation of BCR-ABL1 in CML cells and enabled a quantitative enzymatic analysis of BCR-ABL1 activation. Unexpectedly, we found that the interaction of the SH2 domain with the kinase domain is the critical switch that shifts the BCR-ABL1 activation loop from an otherwise closed to a fully open conformation and enables its autophosphorylation. The activation loop is a central and almost universally used control element that regulates the activity of protein kinases, as the conformation and phosphorylation status of the activation loop determines substrate binding to the active site. In BCR-ABL1, activation loop phosphorylation is required for transformation of fibroblasts and haematopoietic progenitors. We show that the SH2-kinase interaction enables autophosphorylation of the activation loop in trans by rendering a key phosphorylation site (Tyr-412) highly accessible. This requires prior phosphorylation of Tyr-245 in the SH2-kinase linker of BCR-ABL1. Mutational disruption of the SH2-kinase interaction abolished activation loop phosphorylation. Importantly, this effect was independent of the phosphotyrosine binding ability of the SH2 domain, which indicated that the SH2 domain is a true allosteric activator of BCR-ABL1 kinase activity. We also show that the spectrum of tyrosine phosphorylation sites that we mapped by mass spectrometry in vitro were vastly overlapping with the observed BCR-ABL1 phosphorylation sites in CML cells indicating that BCR-ABL1 autophosphorylation might be the major mechanism that determines its cellular phosphorylation status.

In summary, our study demonstrates a novel mechanism by which a protein-protein interaction domain may allosterically mediate the transition of an inactive to an active kinase conformation in a key oncoprotein. This work may serve as an archetype to identify further allosteric regulatory mechanisms in other tyrosine kinases that are activated in haematological malignancies and facilitate the development of new allosteric inhibitors targeting oncogenic tyrosine kinases.

Disclosures

No relevant conflicts of interest to declare.