Identification of the phosphorylation sites on intact TRPM7 channels from mammalian cells.
Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca(2+) and Mg(2+). TRPM7 is ubiquitously expressed in vertebrate cells and contains both an ion channel and a kinase domain. TRPM7 plays an important role in regulating cellular homeostatic levels of Ca(2+) and Mg(2+) in mammalian cells. Although studies have shown that the kinase domain of TRPM7 is required for channel activation and can phosphorylate other target proteins, a systematic analysis of intact TRPM7 channel phosphorylation sites expressed in mammalian cells is lacking. We applied mass spectrometric proteomic techniques to identify and characterize the key phosphorylation sites in TRPM7 channels. We identified 14 phosphorylation sites in the cytoplasmic domain of TRPM7, eight of which have not been previously reported. The identification of phosphorylation sites using antibody-based immunopurification and mass spectrometry is an effective approach for defining the phosphorylation status of TRPM7 channels. The present results show that TRPM7 channels are phosphorylated at multiple sites, which serves as a mechanism to modulate the dynamic functions of TRPM7 channels in mammalian cells.
Department of Physiology, Kyung Hee University School of Medicine, Seoul, South Korea.
Tae Yong
TY
Kim
Seok Kyo
SK
Shin
Min-Young
MY
Song
Ji Eun
JE
Lee
Kang-Sik
KS
Park
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D040901:Proteomics; D050053:TRPM Cation Channels / Q000737:chemistry / Q000378:metabolism*
D050053:TRPM Cation Channels; C421903:Trpm7 protein, mouse