Disruption of Ca²⁺/calmodulin:KSR1 interaction lowers ERK activation

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca²⁺-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca²⁺ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca²⁺/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca²⁺/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca²⁺/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca²⁺/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca²⁺/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe³⁵⁵ with Asp reduces Ca²⁺/CaM binding by 76%. The KSR1-F³⁵⁵D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca²⁺/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca²⁺/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca²⁺ and KSR1-mediated signaling.