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Phosphorylation and regulation of the Na+/H+ exchanger through mitogen-activated protein kinase
H Wang1, N L Silva, P A Lucchesi
1Departments of Biochemistry and Pediatrics, University of Alberta, Edmonton, Canada.
Abstract:
We examined mitogen-activated protein kinase-mediated phosphorylation and activation of the Na+/H+ exchanger isoform type 1. A rabbit skeletal muscle extract was fractionated by FPLC chromatography. Four main fractions had the ability to phosphorylate the carboxyl-terminal region of NHE1. Western blot analysis and immunoprecipitation showed that three of these were associated with MAP kinase-dependent phosphorylation. Phosphorylation studies using purified MAP kinase showed that the region involved was the carboxyl-terminal 178 amino acids of the protein and that the stoichiometry was 1 phosphate/mol of protein. In-gel kinase assays showed that cytosolic extracts from smooth muscle cells also phosphorylate the carboxyl-terminal of NHE1 and that the MAP kinase-dependent phosphorylation could be activated by PDGF and AngII. Mutant cell lines with an inducible dominant negative MAP kinase showed decreased serum activation of Na+/H+ exchange but normal hypertonic activation of the protein. The results show that MAP kinase is intimately involved in regulation of the Na+/H+ exchanger, possibly through phosphorylation of one amino acid of the carboxyl-terminal cytosolic domain.
Insights
Mitogen-activated protein kinase (MAPK) phosphorylates and activates the Na+/H+ exchanger (NHE1). This crucial interaction regulates cellular responses to growth factors and osmotic stress.
Area of Science:
- Cellular Biology
- Molecular Biology
- Physiology
Background:
- The Na+/H+ exchanger isoform type 1 (NHE1) plays a critical role in regulating intracellular pH and cell volume.
- Mitogen-activated protein kinases (MAPKs) are key signaling molecules involved in various cellular processes.
- Understanding the interplay between MAPK and NHE1 is essential for comprehending cellular homeostasis.
Purpose of the Study:
- To investigate the role of MAPK in the phosphorylation and activation of NHE1.
- To identify the specific region of NHE1 targeted by MAPK.
- To elucidate the physiological relevance of MAPK-mediated NHE1 regulation.
Main Methods:
- Fractionation of rabbit skeletal muscle extract using FPLC chromatography.
- Western blot analysis and immunoprecipitation to detect MAPK-associated phosphorylation.
- In-vitro phosphorylation assays using purified MAPK and smooth muscle cell extracts.
- Utilizing mutant cell lines with inducible dominant-negative MAPK.
Main Results:
- Four fractions capable of phosphorylating NHE1's carboxyl-terminal region were identified.
- MAPK was found to be associated with the phosphorylation of NHE1.
- MAPK phosphorylates the carboxyl-terminal 178 amino acids of NHE1 with a stoichiometry of 1 phosphate/mol of protein.
- MAPK-dependent phosphorylation of NHE1 is activated by PDGF and AngII in smooth muscle cells.
- MAPK inhibition reduced serum-stimulated Na+/H+ exchange but not hypertonic-stimulated exchange.
Conclusions:
- MAPK is intimately involved in the regulation of NHE1 activity.
- MAPK-mediated phosphorylation of NHE1 likely occurs at a single amino acid in the carboxyl-terminal cytosolic domain.
- This regulatory pathway is crucial for cellular responses to growth factors and osmotic challenges.