Mass Spectrometric Analysis of TRPM6 and TRPM7 Phosphorylation Reveals Regulatory Mechanisms of the Channel-Kinases
TRPM7 and TRPM6 were the first identified bifunctional channels to contain their own kinase domains,
but how these channel-kinases are regulated is poorly understood. Previous studies identified
numerous phosphorylation sites on TRPM7, but very little is known about TRPM6 phosphorylation
or sites on TRPM7 transphosphorylated by TRPM6. Our mass spectrometric analysis of homomeric
and heteromeric TRPM7 and TRPM6 channels identified phosphorylation sites on both proteins, as
well as several prominent sites on TRPM7 that are commonly modified through autophosphorylation
and transphosphorylation by TRPM6. We conducted a series of amino acid substitution analyses and
identified S1777, in TRPM7’s catalytic domain, and S1565, in TRPM7’s exchange domain that mediates
kinase dimerization, as potential regulatory sites. The phosphomimetic S1777D substitution disrupted
catalytic activity, most likely by causing an electrostatic perturbation at the active site. The S1565D
phosphomimetic substitution also inactivated the kinase but did so without interfering with kinase
dimerization. Molecular modeling indicates that phosphorylation of S1565 is predicted to structurally
affect TRPM7’s functionally conserved N/D loop, which is thought to influence the access of substrate
to the active site pocket. We propose that phosphorylation of S1565 within the exchange domain
functions as a regulatory switch to control TRPM7 catalytic activity.
