Mass Spectrometric Analysis of TRPM6 and TRPM7 Phosphorylation Reveals Regulatory Mechanisms of the Channel-Kinases

2019-03-02T05:55:02Z (GMT) by Na Cai
p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 9.0px Helvetica} <p>TRPM7 and TRPM6 were the first identified bifunctional channels to contain their own kinase domains,</p> <p>but how these channel-kinases are regulated is poorly understood. Previous studies identified</p> <p>numerous phosphorylation sites on TRPM7, but very little is known about TRPM6 phosphorylation</p> <p>or sites on TRPM7 transphosphorylated by TRPM6. Our mass spectrometric analysis of homomeric</p> <p>and heteromeric TRPM7 and TRPM6 channels identified phosphorylation sites on both proteins, as</p> <p>well as several prominent sites on TRPM7 that are commonly modified through autophosphorylation</p> <p>and transphosphorylation by TRPM6. We conducted a series of amino acid substitution analyses and</p> <p>identified S1777, in TRPM7’s catalytic domain, and S1565, in TRPM7’s exchange domain that mediates</p> <p>kinase dimerization, as potential regulatory sites. The phosphomimetic S1777D substitution disrupted</p> <p>catalytic activity, most likely by causing an electrostatic perturbation at the active site. The S1565D</p> <p>phosphomimetic substitution also inactivated the kinase but did so without interfering with kinase</p> <p>dimerization. Molecular modeling indicates that phosphorylation of S1565 is predicted to structurally</p> <p>affect TRPM7’s functionally conserved N/D loop, which is thought to influence the access of substrate</p> <p>to the active site pocket. We propose that phosphorylation of S1565 within the exchange domain</p> <p>functions as a regulatory switch to control TRPM7 catalytic activity.</p>