title: Calmodulin kinase II constitutively binds, phosphorylates, and inhibits brush border Na+/H+ exchanger 3 (NHE3) by a NHERF2 protein-dependent process.
abstract: The epithelial brush border (BB) Na(+)/H(+) exchanger 3 (NHE3) accounts for most renal and intestinal Na(+) absorption. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibits NHE3 activity under basal conditions in intact intestine, acting in the BB, but the mechanism is unclear. We now demonstrate that in both PS120 fibroblasts and polarized Caco-2BBe cells expressing NHE3, CaMKII inhibits basal NHE3 activity, because the CaMKII-specific inhibitors KN-93 and KN-62 stimulate NHE3 activity. This inhibition requires NHERF2. CaMKIIγ associates with NHE3 between aa 586 and 605 in the NHE3 C terminus in a Ca(2+)-dependent manner, with less association when Ca(2+) is increased. CaMKII inhibits NHE3 by an effect on its turnover number, not changing surface expression. Back phosphorylation demonstrated that NHE3 is phosphorylated by CaMKII under basal conditions. This overall phosphorylation of NHE3 is not affected by the presence of NHERF2. Amino acids downstream of NHE3 aa 690 are required for CaMKII to inhibit basal NHE3 activity, and mutations of the three putative CaMKII phosphorylation sites downstream of aa 690 each prevented KN-93 stimulation of NHE3 activity. These studies demonstrate that CaMKIIγ is a novel NHE3-binding protein, and this association is reduced by elevated Ca(2+). CaMKII inhibits basal NHE3 activity associated with phosphorylation of NHE3 by effects requiring aa downstream of NHE3 aa 690 and of the CaMKII-binding site on NHE3. CaMKII binding to and phosphorylation of the NHE3 C terminus are parts of the physiologic regulation of NHE3 that occurs in fibroblasts as well as in the BB of an intestinal Na(+)-absorptive cell.
authors.affiliation: Division of Gastroenterology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Mirza
authors.initials: M
authors.lastname: Zizak
authors.forename: Tiane
authors.initials: T
authors.lastname: Chen
authors.forename: Dorotea
authors.initials: D
authors.lastname: Bartonicek
authors.forename: Rafiquel
authors.initials: R
authors.lastname: Sarker
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Zachos
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.forename: Olga
authors.initials: O
authors.lastname: Kovbasnjuk
authors.forename: Jelena
authors.initials: J
authors.lastname: Korac
authors.forename: Sachin
authors.initials: S
authors.lastname: Mohan
authors.forename: Robert
authors.initials: R
authors.lastname: Cole
authors.forename: Yueping
authors.initials: Y
authors.lastname: Chen
authors.forename: C Ming
authors.initials: CM
authors.lastname: Tse
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008871:Microvilli / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D011522:Protons; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D011522:Protons; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D004815:Epidermal Growth Factor; D012964:Sodium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2