Abstract
The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr416 using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations. We show that the initial steps of kinase activation involve large rearrangements in domain orientation. The kinase domain is highly dynamic and has strong cross-talk with the regulatory domains, which are displaced by autophosphorylation. Although the regulatory domains become more flexible and detach from the kinase domain because of autophosphorylation, the kinase domain gains rigidity, leading to stabilization of the ATP binding site and a 4-fold increase in enzymatic activity. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms.
Highlights
The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events
Tyr416 is part of the activation loop of c-Src, which is located in the C-terminal lobe of the kinase domain (Fig. 1A)
We found that all c-Src constructs were stable, independent of their phosphorylation state (Fig. 2C), but the unfolding cooperativity of the KDP was slightly increased relative to KDU
Summary
The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms. A. S. currently an employee of Leukocare AG, which supports partners in biopharmaceutical development. The tyrosine kinase c-Src is one of nine highly related Src family kinases and the most oncogenic It is ubiquitously expressed in all tissues [6] and plays an important role in numerous signal transduction pathways ranging from cell proliferation to growth and survival. Deregulation of c-Src activity is involved in cancer emergence and progression [7,8,9] and formation of metastases [10, 11]
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