Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel.
Channel-kinase TRPM7/ChaK1 is a member of a recently discovered family of protein kinases called alpha-kinases that display no sequence homology to conventional protein kinases. It is an unusual bifunctional protein that contains an alpha-kinase domain fused to an ion channel. The TRPM7/ChaK1 channel has been characterized using electrophysiological techniques, and recent evidence suggests that it may play a key role in the regulation of magnesium homeostasis. However, little is known about its protein kinase activity. To characterize the kinase activity of TRPM7/ChaK1, we expressed the kinase catalytic domain in bacteria. ChaK1-cat is able to undergo autophosphorylation and to phosphorylate myelin basic protein and histone H3 on serine and threonine residues. The kinase is specific for ATP and cannot use GTP as a substrate. ChaK1-cat is insensitive to staurosporine (up to 0.1 mM) but can be inhibited by rottlerin. Because the kinase domain is physically linked to an ion channel, we investigated the effect of ions on ChaK1-cat activity. The kinase requires Mg(2+) (optimum at 4-10 mM) or Mn(2+) (optimum at 3-5 mM), with activity in the presence of Mn(2+) being 2 orders of magnitude higher than in the presence of Mg(2+). Zn(2+) and Co(2+) inhibited ChaK1-cat kinase activity. Ca(2+) at concentrations up to 1 mM did not affect kinase activity. Considering intracellular ion concentrations, our results suggest that, among divalent metal ions, only Mg(2+) can directly modulate TRPM7/ChaK1 kinase activity in vivo.
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Lillia V
LV
Ryazanova
Maxim V
MV
Dorovkov
Athar
A
Ansari
Alexey G
AG
Ryazanov
D000098:Acetophenones / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D020134:Catalytic Domain; D002412:Cations; D002850:Chromatography, Gel; D003035:Cobalt / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology*; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000502:physiology; D019606:Phosphoamino Acids / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D019311:Staurosporine / Q000494:pharmacology; D050053:TRPM Cation Channels; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D000098:Acetophenones; D001578:Benzopyrans; D002147:Calmodulin; D002412:Cations; D004791:Enzyme Inhibitors; D006657:Histones; D007473:Ion Channels; D007477:Ions; D008565:Membrane Proteins; D004676:Myelin Basic Protein; D019606:Phosphoamino Acids; D050053:TRPM Cation Channels; D013912:Threonine; D003035:Cobalt; D012694:Serine; D004247:DNA; C085746:rottlerin; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C486583:TRPM7 protein, human; D019311:Staurosporine; D008274:Magnesium; D015032:Zinc; D002118:Calcium