TY - JOUR
T1 - Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca2+-activated non-selective cation channel TRPM4
AU - Cerda, Oscar
AU - Cáceres, Mónica
AU - Park, Kang Sik
AU - Leiva-Salcedo, Elías
AU - Romero, Aníbal
AU - Varela, Diego
AU - Trimmer, James
AU - Stutzin, Andrés
PY - 2015/8/24
Y1 - 2015/8/24
N2 - Transient receptor potential melastatin-like 4 (TRPM4) is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.
AB - Transient receptor potential melastatin-like 4 (TRPM4) is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.
KW - Basolateral
KW - Casein kinase
KW - LC-MS/MS
KW - Phosphorylation
KW - TRP channels
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U2 - 10.1007/s00424-014-1610-3
DO - 10.1007/s00424-014-1610-3
M3 - Article
C2 - 25231975
AN - SCOPUS:84937806496
VL - 467
SP - 1723
EP - 1732
JO - Pflugers Archiv European Journal of Physiology
JF - Pflugers Archiv European Journal of Physiology
SN - 0031-6768
IS - 8
ER -