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Expression and phosphorylation of NHE1 in wild‐type and transformed human and rodent fibroblasts
Author(s) -
McSwine Rebecca L.,
Babnigg Gyorgy,
Musch Mark W.,
Chang Eugene B.,
Villereal Mitchel L.
Publication year - 1994
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041610220
Subject(s) - sodium–hydrogen antiporter , phosphorylation , microbiology and biotechnology , cell culture , cytoplasm , biology , fibroblast , embryonic stem cell , messenger rna , chemistry , gene , biochemistry , genetics , organic chemistry , sodium
Activation and phosphorylation of the Na + /H + exchanger occurs with a diverse group of mitogens such as phorbol myristate acetate (PMA), epidermal growth factor (EGF), thrombin and serum (Sardet et al., 1990, Science, 247:723‐‐726). Some of these growth factors have been shown to differentially activate the Na + /H + exchanger in various fibroblasts (Etscheid et al., 1990, Am. J. Physiol., 259 :C549‐‐C556; Muldoon, L. L., et al., 1987, Am. J. Physiol., 253 :C219‐‐C229). However, alterations in the expression and phosphorylation of NHE1 in various fibroblasts has not been examined with respect to a potential mechanism of differential activation of the exchanger. To pursue this question, a novel antibody, anti‐XB 17 , directed to the cytoplasmic tail of NHE1 was characterized and then utilized to examine the expression of NHE1 protein and the level of phosphorylation of the serum stimulated exchanger in human embryonic lung fibroblasts (WI‐38), SV40‐transformed WI‐38, and nontransformed human foreskin fibroblasts (HSWP) cells. The level of mRNA expressed in these cells was also examined. Results indicate that the parental cell lines and other nontransformed fibroblasts express NHE1. Although the transformed cell lines express NHE1 mRNA in approximately similar abundance to the parental lines, they contain decreased quantity of NHE1 exchanger/mg membrane protein as recognized by anti‐XB 17 Ab. The mechanism that results in the apparent decrease in NHE1 protein levels in the transformed cells is not known. Also, the SV40‐transformed cells express and exchanger with a higher apparent molecular weight. The WI‐38 cells demonstrate phosphorylation of NHE1 in response to mitogenic stimulation. Although the nontransformed HSWP cells have a high level of Na + /H + exchanger protein, they do not show a significant increase in phosphorylation following serum stimulation, when examined by immunoprecipitation, and analysis on 1‐D gels. However, subsequent studies of tryptic phosphopeptides from the immunoprecipitated exchanger reveal that serum‐stimulated phosphorylation of one tryptic peptide does occur but may be masked in the first dimension by differential phosphorylation of other tryptic peptides that are more heavily phosphorylated in unstimulated cells. © 1994 Wiley‐Liss, Inc.