Abstract
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.
Highlights
TRPM7 and its close homologue TRPM6 are bifunctional proteins consisting of a cation-permeating channel with a COOH-terminal functional kinase domain
As technological advances have improved the sensitivity for identifying phosphorylation sites on proteins, we were motivated to discover sites that may have been missed in previous analyses of TRPM7, as well as to identify phosphorylation sites on TRPM6, whose phosphorylation has not yet been systematically investigated
Unlike what was previously reported for purified COOH-terminal fragment of TRPM7, where “massive” autophosphorylation was observed[24], our mass spectrometric analysis identified more selective in vivo phosphorylation of full-length TRPM7 in the absence of ATP stimulation (Fig. 2)
Summary
TRPM7 and its close homologue TRPM6 are bifunctional proteins consisting of a cation-permeating channel with a COOH-terminal functional kinase domain. The kinase domain of TRPM7 assembles into functional dimers through the exchange of a short stretch of amino acids NH2-terminal to the core catalytic domain[17]. Unlike other members of the alpha-kinase family and conventional protein kinases, TRPM7 has been shown to undergo extensive autophosphorylation within a serine/threonine-rich region proximal to the exchange domain of the kinase and the kinase’s catalytic core[24,25]. Studies of other members of the alpha-kinases family such as eEF2K and Dictyostelium MHCKs identified important autophosphorylation sites outside the catalytic core that regulate kinase activity[26,27]; comparable regulatory phosphorylation sites have not been determined for TRPM7 or TRPM6. Our results point to a critical role for phosphorylation in controlling the catalytic activity of the channel-kinases
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