JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro.
نویسندگان
چکیده
Cell shrinkage phosphorylates and activates the Na-K-2Cl cotransporter (NKCC1), indicating the presence of a volume-sensitive protein kinase. To identify this kinase, extracts of normal and shrunken aortic endothelial cells were screened for phosphorylation of NKCC1 fusion proteins in an in-the-gel kinase assay. Hypertonic shrinkage activated a 46-kDa kinase that phosphorylated an NH2-terminal fusion protein, with weaker phosphorylation of a COOH-terminal fusion protein. This cytosolic kinase was activated by both hypertonic and isosmotic shrinkage, indicating regulation by cell volume rather than osmolarity. Subsequent studies identified this kinase as c-Jun NH2-terminal kinase (JNK). Immunoblotting revealed increased JNK activity in shrunken cells; there was volume-sensitive phosphorylation of NH2-terminal c-Jun fusion protein; immunoprecipitation of JNK from shrunken cells but not normal cells phosphorylated NKCC1 in gel kinase assays; and treatment of cells with tumor necrosis factor, a known activator of JNK, mimicked the effect of hypertonicity. We conclude that JNK is a volume-sensitive kinase in endothelial cells that phosphorylates NKCC1 in vitro. This is the first demonstration of a volume-sensitive protein kinase capable of phosphorylating a volume-regulatory transporter.
منابع مشابه
Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride.
Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl(-) transport. Reductions in either cell volume or cytosolic Cl(-) concentration ([Cl(-)](i)) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl(-)](i) was evaluated in m...
متن کاملActivation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.
The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7...
متن کاملNa+-K+-2Cl- cotransporter in immature cortical neurons: A role in intracellular Cl- regulation.
Na+-K+-2Cl- cotransporter has been suggested to contribute to active intracellular Cl- accumulation in neurons at both early developmental and adult stages. In this report, we extensively characterized the Na+-K+-2Cl- cotransporter in primary culture of cortical neurons that were dissected from cerebral cortex of rat fetus at embryonic day 17. The Na+-K+-2Cl- cotransporter was expressed abundan...
متن کاملCharacterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter.
Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We sp...
متن کاملNa+-K+-2Cl-cotransport in Ehrlich cells: regulation by protein phosphatases and kinases.
To identify protein kinases (PK) and phosphatases (PP) involved in regulation of the Na+-K+-2Cl-cotransporter in Ehrlich cells, the effect of various PK and PP inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin A (Cal-A) was a potent activator of Na+-K+-2Cl-cotransport (EC50 = 35 nM). Activation by Cal-A was rapid (<1 min) but transient. Inactivation is probably due to a 10%...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The American journal of physiology
دوره 277 3 Pt 1 شماره
صفحات -
تاریخ انتشار 1999