A Dominant Negative Mutant of the KCC1 K-Cl Cotransporter

With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed cotemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

Section: Fp Probes Of Nkcc1-supporting

confidence: 76%

Section: Fp Probes Of Nkcc1-mentioning

confidence: 99%

See 1 more Smart Citation

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

Pedersen¹,

Carmosino

Forbush

2008

“…9). This dominantnegative effect of inactive NKCCs has been noted in other mutagenesis studies in our laboratory (5) and has also been seen with a truncation variant of NKCC2 (24), in inactive mutants of KCC1 (25), and in an interaction between human CIP and NKCC2 (4). At this point, it is unclear whether the present effects are indicative of functional protein-protein interaction at the plasma membrane, competition for an unknown cellular component, or overexpression bottlenecks in the synthesis and delivery of NKCC.…”

Section: Resultsmentioning

confidence: 54%

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Darman

Forbush

2002

219

221

The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylationdependent mechanism. We found a phosphorylation stoichiometry of 3.0 ؎ 0.4 phosphorylated residues/ NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr 184 , Thr 189 , and Thr 202 ) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. The secretory Na-K-Cl cotransporter NKCC1 is a major pathway for net influx of Cl Ϫ in many cells (1, 2). Along with the absorptive Na-K-Cl cotransporter NKCC2 and the thiazidesensitive Na-Cl transporter NCC (or TSC), NKCC1 belongs to the sodium-coupled branch of the cation chloride cotransporter family; this family also includes the K-Cl transporters (KCCs) (3) and two other major branches of putative transporters, including human CIP (4) and SLC12-8 (GenBank TM /EBI accession number AAK94307), whose transport functions are unknown. These proteins are all predicted to have 12 transmembrane-spanning domains that are responsible for ion transport properties (5) and large cytoplasmic N and C termini that are candidates for regulatory domains. NKCC1 is expressed in many cell types and is involved in the regulation of both cell volume and intracellular Cl Ϫ concentration (2, 6). This isoform is also a major component of the basolateral membrane of secretory cells, mediating Cl Ϫ influx, the first step in the transepithelial movement of Cl Ϫ . In contrast, the NKCC2 isoform is limited to the apical membranes of absorptive epithelia such as the thick ascending limb of Henle's loop, where it is a major determinant of NaCl reabsorption from the tubular fluid.In order that the processes of regulatory volume increase, secretion, and absorption may be tightly controlled, the Na-K-Cl cotransporter is subject to strict regulation. The cotransporter is inactive in the basolateral membranes of secretory cells until the application of secretagogues or cell shrinkage causes a phosphorylation-dependent activation of the cotransporter (7-10). Studies on shark rectal gland secretory epithelia have shown an increase in phosphorylation at serines and threonines in response to forskolin or hypertonic stress, and the N terminus of NKCC1 has been shown to be the locus of at least some of the phosphoregulatory sites (7, 11). Although, in many cells, PKA 1 agonists effect an increase in Na-K-Cl co...

“…The removal of 117 amino acids from the N-terminal cytoplasmic domain of mouse KCC1 (KCC1 ⌬N117) confers a dominantnegative phenotype when co-expressed with wild-type KCC1, KCC3, or KCC4 polypeptides (9,12). The dominant-negative mouse KCC1 ⌬N117 cDNA was subcloned into the eukaryotic expression vector pCDNA3 (Invitrogen), transfected into SiHa and OVCAR-3 cell lines by lipofection, and stable lines were selected as described previously (9).…”

Section: Cell Cultures and Creation Of Kcc Mutant Cell Lines-mentioning

confidence: 99%

Insulin-like Growth Factor 1 Stimulates KCl Cotransport, Which Is Necessary for Invasion and Proliferation of Cervical Cancer and Ovarian Cancer Cells

Shen¹,

Lin²,

Hsu³

et al. 2004

Self Cite

The mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with membrane ion transport system to modulate epithelial cell motility and proliferation remain poorly understood. Here, we investigated the role of electroneutral KCl cotransport (KCC), in IGF-1-dependent invasiveness and proliferation of cervical and ovarian cancer cells. IGF-1 increased KCC activity and mRNA expression in a dose-and time-dependent manner in parallel with the enhancement of regulatory volume decrease. IGF-1 treatment triggers phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascades leading to the activation of Akt and extracellular signal-regulated kinase1/2 (Erk1/2), respectively. The activated Erk1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways are differentially required for IGF-1-stimulated biosynthesis of KCC polypeptides. Specific reduction of Erk1/2 protein levels with small interference RNA abolishes IGF-1-stimulated KCC activity. Pharmacological inhibition and genetic modification of KCC activity demonstrate that KCC is necessary for IGF-1-induced cancer cell invasiveness and proliferation. IGF-1 and KCC colocalize in the surgical specimens of cervical cancer (n ‫؍‬ 28) and ovarian cancer (n ‫؍‬ 35), suggesting autocrine or paracrine IGF-1 stimulation of KCC production. Taken together, our results indicate that KCC activation by IGF-1 plays an important role in IGF-1 signaling to promote growth and spread of gynecological cancers.