%0 Journal Article %1 Iwam_1998_17230 %A Iwamoto, T. %A Pan, Y. %A Nakamura, T. Y. %A Wakabayashi, S. %A Shigekawa, M. %D 1998 %J Biochemistry %K Acetate, Acid Amino Animals; C, Calcium Cell Cricetinae; Cricetulus; Data; Dogs; Exchanger, Fluid, Intracellular Isoforms, Kinase Line; Membrane Molecular Mutagenesis, Phosphorylation, Protein Proteins; Radioisotopes, Rats; Sequence Sequence; Site-Directed; Sodium-Calcium Tetradecanoylphorbol Transport biosynthesis/genetics/metabolism; drug effects; metabolism/physiology; metabolism; pharmacology %N 49 %P 17230--17238 %R 10.1021/bi981521q %T Protein kinase C-dependent regulation of Na$^+$/Ca$^2+$ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation. %U http://dx.doi.org/10.1021/bi981521q %V 37 %X We compared the phosphorylation-dependent regulation of three mammalian Na$^+$/Ca$^2+$ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na$^+$i-dependent 45Ca$^2+$ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using 32Porthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.

@article{Iwam_1998_17230, abstract = {We compared the phosphorylation-dependent regulation of three mammalian {N}a$^{+}$/{C}a$^{2+}$ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. {N}a$^{+}$i-dependent 45{C}a$^{2+}$ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Iwamoto, T. and Pan, Y. and Nakamura, T. Y. and Wakabayashi, S. and Shigekawa, M.}, biburl = {https://www.bibsonomy.org/bibtex/2bd2501d28a886216dfd4c37101955664/hake}, description = {The whole bibliography file I use.}, doi = {10.1021/bi981521q}, file = {Iwam_1998_17230.pdf:Iwam_1998_17230.pdf:PDF}, institution = {Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.}, interhash = {8071adcf44c5095928f927966a34044d}, intrahash = {bd2501d28a886216dfd4c37101955664}, journal = {Biochemistry}, keywords = {Acetate, Acid Amino Animals; C, Calcium Cell Cricetinae; Cricetulus; Data; Dogs; Exchanger, Fluid, Intracellular Isoforms, Kinase Line; Membrane Molecular Mutagenesis, Phosphorylation, Protein Proteins; Radioisotopes, Rats; Sequence Sequence; Site-Directed; Sodium-Calcium Tetradecanoylphorbol Transport biosynthesis/genetics/metabolism; drug effects; metabolism/physiology; metabolism; pharmacology}, month = Dec, number = 49, pages = {17230--17238}, pdf = {Iwam_1998_17230.pdf}, pii = {bi981521q}, pmid = {9860837}, timestamp = {2009-06-03T11:21:16.000+0200}, title = {Protein kinase C-dependent regulation of {N}a$^{+}$/{C}a$^{2+}$ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation.}, url = {http://dx.doi.org/10.1021/bi981521q}, volume = 37, year = 1998 }