Moriah J Hovde1, Danielle E Bolland2, Aryna Armand3, Emily Pitsch4, Clare Bakker5, Amanda J Kooiker6, Joseph J Provost7, Roxanne A Vaughan8, Mark A Wallert9, James D Foster10. 1. Department of Biomedical Sciences, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND 58202, United States of America. Electronic address: Moriah.hovde@mgh.harvard.edu. 2. Department of Biomedical Sciences, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND 58202, United States of America. Electronic address: dbolland@morris.umn.edu. 3. Department of Chemistry and Biochemistry, University of San Diego, San Diego, CA 92110, United States of America. Electronic address: aarmand@sandiego.edu. 4. Department of Chemistry and Biochemistry, University of San Diego, San Diego, CA 92110, United States of America. 5. Department of Chemistry and Biochemistry, University of San Diego, San Diego, CA 92110, United States of America. Electronic address: cbakker@sandiego.edu. 6. Biology Department, Bemidji State University, Bemidji, MN 56601, United States of America. Electronic address: Amanda.kooiker@live.bemidjistate.edu. 7. Department of Chemistry and Biochemistry, University of San Diego, San Diego, CA 92110, United States of America. Electronic address: josephprovost@sandiego.edu. 8. Department of Biomedical Sciences, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND 58202, United States of America. Electronic address: roxanne.vaughan@UND.edu. 9. Biology Department, Bemidji State University, Bemidji, MN 56601, United States of America. Electronic address: mark.wallert@bemidjistate.edu. 10. Department of Biomedical Sciences, University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND 58202, United States of America. Electronic address: james.d.foster@UND.edu.
Abstract
AIMS: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. MAIN METHODS: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. KEY FINDINGS: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SIGNIFICANCE: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.
AIMS: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. MAIN METHODS: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. KEY FINDINGS: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SIGNIFICANCE: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.
Authors: Ivana Mikic; Sonia Planey; Jun Zhang; Carolina Ceballos; Terri Seron; Benedikt von Massenbach; Rachael Watson; Scott Callaway; Patrick M McDonough; Jeffrey H Price; Edward Hunter; David Zacharias Journal: Methods Enzymol Date: 2006 Impact factor: 1.600