The crosstalk between the Ubiquitin and the Calcium regulatory pathways

Calcium and ubiquitin (Ub) signals are decoded by Calmodulin (CaM) and Ub-receptors (respectively) and modulate numerous cellular pathways. We identified a novel crosstalk between the Ub and Ca²⁺ signaling pathways. Although CaM was reported to undergo ubiquitylation in the 80s, this is the first report for a ligase that ubiquitylates it. Using our E. coli based selection system (Nature methods 2016) and the tight correlation between ubiquitylation and bacterial growth (Nature communication 2016) we found that CaM is targeted by one of our studied ligases. Interestingly, the two CaM lobes interact with the ligase in a Ca²⁺ dependent manner. Mutations analyses showed that at low Ca²⁺ concentrations either the N- or the C-lobe interact and promote CaM ubiquitylation. However, at high Ca²⁺ concentrations the lobes simultaneously interact with the ligase and significantly inactivate it. We are now assessing a structural model for this substrate/inhibitor mechanism. Recently, we discovered and characterized in detail a novel allosteric mechanism that regulates NEDD4 by self-ubiquitylation (EMBO 2017). Structural insights into this allosteric mechanism facilitated the design of an unregulated (hyperactive) ligase mutant. This mutant presents a physiological phenotype in response to changes in Ca²⁺ concentration in mammalian cells. Our data indicate that the ligase regulates the level of semi Ca²⁺-occupied CaM, hence accelerates the titration of CaM by Ca²⁺ bursts and expedites the response to Ca²⁺ signaling. The function of the selection system in drug and PROTAC discovery with Ubox/RINGs, RBRs, HECTs and CRLs and their cognate targets (including Ca²⁺ related) will be presented.