Protein phosphatase regulation of Na+/H+ exchanger isoform I.
We investigated regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) by dephosphorylation. Treatment of primary cultures of cardiomyocytes with the phosphatase inhibitor okadaic acid increased the rate of recovery from an acid load, suggesting that the okadaic acid sensitive PP1 may be involved in NHE1 regulation in vivo. We examined the ability of purified protein phosphatases PP1, PP2A, and PP2B to dephosphorylate the regulatory cytoplasmic tail. NHE1 was completely dephosphorylated by PP1, poorly dephosphorylated by PP2A, and not dephosphorylated by PP2B. Examination of NHE1 binding to PP1 or PP2B revealed that an association occurs between NHE1 and PP1 both in vitro and in vivo, but NHE1 did not associate with full-length PP2B. We expressed PP1 or inhibitor 2, a specific PP1 inhibitor, in cell lines to examine the effect of PP1 on NHE1 activity in vivo. Overexpression of PP1 causes a decrease in NHE1 activity but does not affect stimulation by thrombin. Cell lines expressing the specific PP1 inhibitor, inhibitor 2, had elevated proton efflux rates and could not be further stimulated by the Na(+)/H(+) exchanger agonist thrombin. The results suggest that PP1 is an important regulatory phosphatase of NHE1, that it can bind to and dephosphorylate the protein, and that it regulates NHE1 activity in vivo.
Misik
Angelika J
AJ
Department of Biochemistry, Faculty of Medicine, CIHR Membrane Protein Research Group, University of Alberta, 347 Medical Science Building, Edmonton, Alberta, Canada T6G 2H7.
Perreault
Kathleen
K
Holmes
Charles F B
CF
Fliegel
Larry
L
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
D027682:Cation Transport Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D019319:Okadaic Acid; D004247:DNA; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases