Massive Autophosphorylation of the Ser/Thr-Rich Domain Controls Protein Kinase Activity of TRPM6 and TRPM7

被引:78
作者
Clark, Kristopher [1 ]
Middelbeek, Jeroen [2 ]
Morrice, Nick A. [4 ]
Figdor, Carl G. [1 ]
Lasonder, Edwin [3 ]
van Leeuwen, Frank N. [1 ,2 ]
机构
[1] Radboud Univ Nijmegen, Med Ctr, Nijmegen Ctr Mol Life Sci, Dept Tumor Immunol, NL-6525 ED Nijmegen, Netherlands
[2] Radboud Univ Nijmegen Med Ctr, Med Ctr, Nijmegen Ctr Mol Life Sci, Lab Pediat Oncol, Nijmegen, Netherlands
[3] Radboud Univ Nijmegen Med Ctr, Nijmegen Ctr Mol Life Sci, Ctr Mol & Biomol Informat, Nijmegen, Netherlands
[4] Univ Dundee, James Black Ctr, MRC Protein Phosphorylat Unit, Dundee, Scotland
来源
PLOS ONE | 2008年 / 3卷 / 03期
关键词
D O I
10.1371/journal.pone.0001876
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical alpha-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 phosphorylation. 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.
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页数:10
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