TY - JOUR
T1 - Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7
AU - Clark, Kristopher
AU - Middelbeek, Jeroen
AU - Morrice, Nick A.
AU - Figdor, Carl G.
AU - Lasonder, Edwin
AU - van Leeuwen, Frank N.
N1 - Funding information: The work was supported by a short-term fellowship from FEBS to KC and grants from the Dutch Cancer Society to FvL (KUN 2002-2593/KUN 2007-
3733).
PY - 2008/3/26
Y1 - 2008/3/26
N2 - TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical α-kinase domain. While the gating properties of TRPM6 and TRPM7 channels have been studied in detail, little is known about the mechanisms regulating kinase activity. Recently, we found that TRPM7 associates with its substrate myosin II via a kinase-dependent mechanism suggesting a role for autophosphorylation in substrate recognition. Here, we demonstrate that the cytosolic C-terminus of TRPM7 undergoes massive autophosphorylation (32±4 mol/mol), which strongly increases the rate of substrate phosporylation. Phosphomapping by mass spectrometry indicates that the majority of autophosphorylation sites (37 out of 46) map to a Ser/Thr-rich region immediately N-terminal of the catalytic domain. Deletion of this region prevents substrate phosphorylation without affecting intrinsic catalytic activity suggesting that the Ser/Thr-rich domain contributes to substrate recognition. Surprisingly, the TRPM6-kinase is regulated by an analogous mechanism despite a lack of sequence conservation with the TRPM7 Ser/ Thr-rich domain. In conclusion, our findings support a model where massive autophosphorylation outside the catalytic domain of TRPM6 and TRPM7 may facilitate kinase-substrate interactions leading to enhanced phosphorylation of those substrates.
AB - TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical α-kinase domain. While the gating properties of TRPM6 and TRPM7 channels have been studied in detail, little is known about the mechanisms regulating kinase activity. Recently, we found that TRPM7 associates with its substrate myosin II via a kinase-dependent mechanism suggesting a role for autophosphorylation in substrate recognition. Here, we demonstrate that the cytosolic C-terminus of TRPM7 undergoes massive autophosphorylation (32±4 mol/mol), which strongly increases the rate of substrate phosporylation. Phosphomapping by mass spectrometry indicates that the majority of autophosphorylation sites (37 out of 46) map to a Ser/Thr-rich region immediately N-terminal of the catalytic domain. Deletion of this region prevents substrate phosphorylation without affecting intrinsic catalytic activity suggesting that the Ser/Thr-rich domain contributes to substrate recognition. Surprisingly, the TRPM6-kinase is regulated by an analogous mechanism despite a lack of sequence conservation with the TRPM7 Ser/ Thr-rich domain. In conclusion, our findings support a model where massive autophosphorylation outside the catalytic domain of TRPM6 and TRPM7 may facilitate kinase-substrate interactions leading to enhanced phosphorylation of those substrates.
UR - http://www.scopus.com/inward/record.url?scp=46749143291&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0001876
DO - 10.1371/journal.pone.0001876
M3 - Article
C2 - 18365021
AN - SCOPUS:46749143291
SN - 1932-6203
VL - 3
JO - PLoS One
JF - PLoS One
IS - 3
M1 - e1876
ER -