title: Identification of dimer interactions required for the catalytic activity of the TRPM7 alpha-kinase domain.
abstract: TRPM7 (transient receptor potential melastatin) combines an ion channel domain with a C-terminal protein kinase domain that belongs to the atypical alpha-kinase family. The TRPM7 alpha-kinase domain assembles into a dimer through the exchange of an N-terminal segment that extends from residue 1551 to residue 1577 [Yamaguchi, Matsushita, Nairn and Kuriyan (2001) Mol. Cell 7, 1047-1057]. Here, we show, by analysis of truncation mutants, that residues 1553-1562 of the N-terminus are essential for kinase activity but not dimer formation. Within this 'activation sequence', site-directed mutagenesis identified Tyr-1553 and Arg-1558 as residues critical for activity. Examination of the TRPM7 kinase domain structure suggests that the activation sequence interacts with the other subunit to help position a catalytic loop that contains the invariant Asp-1765 residue. Residues 1563-1570 of the N-terminal segment are critical for dimer assembly. Mutation of Leu-1564, Ile-1568 or Phe-1570 to alanine abolished both kinase activity and dimer formation. The activity of a monomeric TRPM7 kinase domain lacking the entire N-terminal segment was rescued by a GST (glutathione transferase) fusion protein containing residues 1548-1576 of TRPM7, showing that all interactions essential for activity are provided by the N-terminal segment. Activity was also restored by GST fused to the N-terminal segment of TRPM6 (residues 1711-1740), demonstrating the feasibility of forming functional TRPM6-TRPM7 alpha-kinase domain heterodimers. It is proposed that covalent modifications or binding interactions that alter the conformation of the N-terminal exchanged segment may provide a means to regulate TRPM7 kinase activity.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Crawley
authors.forename: Graham P
authors.initials: GP
authors.lastname: Côté
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D051379:Mice; D011494:Protein Kinases / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D050053:TRPM Cation Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D050053:TRPM Cation Channels; D011494:Protein Kinases; C421903:Trpm7 protein, mouse