Characterization of a Novel Interaction between the Secretory Na+-K+-Cl- Cotransporter and the Chaperone hsp90

Abstract

The first isoform of the Na ؉ -K ؉ -Cl ؊ cotransporter (NKCC1) is of central importance for the control of cellular ion concentration and epithelium-mediated salt secretion. Several studies have established that a change in intracellular [Cl ؊ ] (Cl ؊ i ) represents a key signaling mechanism by which NKCC1-induced Cl ؊ movement is autoregulated and by which Cl ؊ entry and exit on opposite sides of polarized cells are coordinated. Although this signaling mechanism is coupled to a pathway that leads to post-translational modification of the carrier, no unifying model currently accounts for the ion dependence of NKCC1 regulation. In this paper, evidence is presented for the first time that hsp90 associates with the cytosolic C terminus of NKCC1, probably when the carrier is predominantly in its unfolded form during early biogenesis. Evidence is also presented that the Cl ؊ i -dependent regulatory pathway can be activated by a thermal stress but that it is no longer operational if NKCC1-expressing cells are pretreated with geldanamycin, an antibiotic that inhibits hsp90, albeit nonspecifically. Taken together, our data indicate that binding of hsp90 to NKCC1 may be required for Na؊ cotransport to occur at the cell surface and that it could play an important role in ion-dependent signaling mechanisms, insofar as the maneuvers that were used to alter the expression or activity of the chaperone do not exert their main effect by inducing other cellular events such as the unfolded protein response. Further studies will be required to elucidate the functional relevance of this novel interaction. Cation-ClϪ cotransporters (CCCs) 1 are polytopic membrane proteins that couple the movement of Cl Ϫ to that of Na ϩ and/or K ϩ ions in or out of cells (1-4). Seven such proteins have been identified to date; the Na ϩ -K ϩ -Cl Ϫ cotransporters (NKCCs) isoform 1 and 2 (5, 6), the Na ϩ -Cl Ϫ cotransporter isoform 1 (7), and the K ϩ -Cl Ϫ cotransporters isoform 1-4 (8 -11). Other proteins that are homologous to the CCCs have also been identified (12); they are termed orphan CCCs because their ionic substrates have not been identified to date.The NKCC1 is considered a housekeeping carrier that is responsible for the bumetanide-sensitive component of Na ϩ -K ϩ -Cl Ϫ cotransport across cellular membranes (1-4). One of its main functions is to regulate cell volume (V cell ) and intracellular ClBecause of its basolateral localization in polarized cells, this carrier also promotes fluid and electrolyte secretion through a variety of epithelia by cooperating with apically disposed ion transport systems including Cl Ϫ channels (13-17). Several lines of evidence suggest that NKCC1 activity is increased by phosphorylation of its cytosolic termini (see Fig. 1) in response to Cl Ϫ i reduction or cell shrinkage. To this effect, recent studies have reported binding of proline-alanine-rich stress-related kinase (PASK) to NKCC1 and provided strong evidence that this interaction leads to phosphorylation-dependent activation of Na 18 -20). Along the sa...

Direct control of Na+-K+-2Cl−-cotransport protein (NKCC1) expression with aldosterone

“…Previous studies implicate posttranslational phosphorylation associated with NKCC1 expression changes (1,9,23,25,29,44,50). In contrast, the present investigation is the first report that ALD exerts its effects on NKCC1 expression via prevention of posttranslational ubiquitination, e.g., reduces proteasome-dependent degradation of the NKCC1 protein.…”

“…Mutation of this domain induces a kidney disease called Liddle's syndrome. Since NKCC1 and NCCs belong to the cation Cl Ϫ cotransporters (CCCs), and the CCCs exhibit a common structure in their functional regulation domains, such as the membrane associated domain and the phosphoacceptor sites (42,50), we hypothesize that there is a similarity in the regulation of NCC and NKCC1 inductions by ALD. To test this, we found that ALD increases the phosphorylation of SGK1 and Nedd4 -2 and concomitantly there is an increased induction of NKCC1 protein expression (Fig.…”

NKCC2 Surface Expression in Mammalian Cells

“…Such mechanisms have been reported for the basolateral isoform, NKCC1. Proteins that bind to NKCC1 include SPAK and OSR1, PP1, PP2A, CIP, and Hsp90 (12)(13)(14)(15)(16)(17). The identification of several NKCC1-binding proteins supported further the view that regulation of NKCC2 might also involve a network of protein-protein interactions.…”

Chloride Is Essential for Capacitation and for the Capacitation-associated Increase in Tyrosine Phosphorylation

“…This later transporter family is composed of three subfamilies with a total of seven members (37,38): one Na ϩ / Cl Ϫ cotransporter (NCC), which can be inhibited by diuretics such as hydrochlorothiazide; two NKCCs, inhibited by bumetanide and less potently by furosemide; and four Na ϩ -independent K ϩ /Cl Ϫ cotransporters (KCC), which can be specifically blocked by R(ϩ)-butylindazone (39). No effects were observed on tyrosine phosphorylation ( Fig.…”