Oxidative stress-responsive kinase (OSR) 1 and sterile20-related, proline-, alanine-rich kinase (SPAK) are Ste20p-related protein kinases that bind to the sodium, potassium, two chloride cotransporter, NKCC. Here we present evidence that the protein kinase with no lysine [K] (WNK) 1 regulates OSR1, SPAK, and NKCC activities. OSR1 exists in a complex with WNK1 in cells, is activated by recombinant WNK1 in vitro, and is phosphorylated in a WNK1-dependent manner in cells. Depletion of WNK1 from HeLa cells by using small interfering RNA reduces OSR1 kinase activity. In addition, depletion of either WNK1 or OSR1 reduces NKCC activity, indicating that WNK1 and OSR1 are both required for NKCC function. OSR1 and SPAK are likely links between WNK1 and NKCC in a pathway that contributes to volume regulation and blood pressure homeostasis in mammals.blood pressure ͉ kinase ͉ osmotic ͉ stress P rotein kinase cascades mediate cellular responses to extracellular signals and environmental change. A large group of protein kinases with remarkably complex and diverse functions are related to the yeast protein kinase Ste20p (1). Ste20p was identified as a component of a mitogen-activated protein kinase cascade in the yeast pheromone-induced mating pathway (2, 3). One substrate of Ste20p is Ste11p, the MAP kinase kinase kinase in the module; thus, Ste20p is the prototypical MAP4K. Ste20p also controls cytoskeletal reorganization required for budding in yeast. Ste20p kinases coordinate the activities of downstream molecules not only because of their catalytic activities but also because of their capacities to bind and organize upstream molecules, including receptors and adaptors, and downstream molecules, including cytoskeletal elements and other protein kinases.More than 30 Ste20p-related kinases are encoded in the human genome in two structurally distinct subfamilies, the p21-activated kinase (PAK) subfamily and the germinal center kinase (GCK) subfamily (1). The PAKs bind to GTP-liganded forms of the Rho family small G proteins Rac and Cdc42 and are key regulators of cytoskeletal organization and cell motility (4). The germinal center kinase-related kinases all contain N-terminal catalytic domains and diverse C-terminal domains that were used to categorize them into eight subfamilies (1).OSR1 and the sterile20-related, proline-, alanine-rich kinase (SPAK) are the only two mammalian protein kinases in the germinal center kinase-VI subfamily. Although it has not been shown to be sensitive to oxidative stress, OSR1 was named for its similarity to oxidative stress-responsive kinase SOK1 (Ste20͞ oxidant stress response kinase-1) (5). OSR1 does respond to osmotic stress and phosphorylates PAK1, inhibiting its responsiveness to Cdc42 (6). In addition to their similar kinase domains, OSR1 and SPAK share two conserved C-terminal regions, known as PF1 and PF2 domains. The PF1 domain lies immediately C-terminal to the protein kinase domain and is required for catalytic activity of OSR1, although not part of the consensus core of protein kin...

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast twohybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a Cterminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.WNKs (With No lysine (K)) are serine/threonine protein kinases implicated in regulating ion permeability in epithelia (1-4). There are four mammalian WNK family members (5, 6), and mutations in two of them, WNK1 and WNK4, have been linked to a hereditary form of human hypertension known as pseudohypoaldosteronism type II (1). This discovery has provoked a broad search for physiological mechanisms by which WNKs regulate blood pressure, as well as relationships between mutations in WNKs and other types of hypertension (7,8). Mutations in WNK4 are in the coding sequence, whereas mutations in WNK1 are intronic and cause overexpression of the wild-type protein (1). Because we originally isolated cDNAs encoding WNK1 (5), we have been working to understand signal transduction events mediated by WNK1 and the biochemical mechanisms utilized by it that might modulate membrane permeability (9, 10).WNKs are unique because the lysine required for phosphoryl transfer lies in the phosphate anchor ribbon (kinase subdomain I) instead of ␤ strand 3 (kinase subdomain II), its position in all other members of the protein kinase superfamily (5). The kinase domains of WNKs are located near their N termini. WNKs contain a conserved autoinhibitory domain, first identified in WNK1, and two predicted coiled-coil domains, which are located C-terminal to the kinase domain (6, 9, 11). The WNK1 autoinhibitory domain (residues 485-555) reduces WNK1 autophosphorylation and substrate phosphorylation (9). Similarly, the WNK4 autoinhibitory domain (residues 444 -518) was reported to inhibit WNK1 autophosphorylation, suggesting WNKs may modulate the kinase activity of other family members (11).In this study, we have continued to characterize the biochemical properties and regulation of WNK1 that may ...

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Fulllength and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM.

MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.

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5-bisphosphate (PIP 2) affects profoundly several cardiac ion channels and transporters, and studies of PIP2-sensitive currents in excised patches suggest that PIP 2 can be synthesized and broken down within 30 s. To test when, and if, total phosphatidylinositol 4-phosphate (PIP) and PIP2 levels actually change in intact heart, we used a new, nonradioactive HPLC method to quantify anionic phospholipids. Total PIP and PIP2 levels (10-30 mol/kg wet weight) do not change, or even increase, with activation of G␣ q/phospholipase C (PLC)-dependent pathways by carbachol (50 M), phenylephrine (50 M), and endothelin-1 (0.3 M). Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1M) both cause 30% reduction of PIP2 in ventricles, suggesting that diacylglycerol (DAG)-dependent mechanisms negatively regulate cardiac PIP2. PIP2, but not PIP, increases reversibly by 30% during electrical stimulation (2 Hz for 5 min) in guinea pig left atria; the increase is blocked by nickel (2 mM). Both PIP and PIP2 increase within 3 min in hypertonic solutions, roughly in proportion to osmolarity, and similar effects occur in multiple cell lines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP2 metabolism might be sensitive to membrane tension, per se. phosphatidylinositol 4,5-bisphosphate; phosphatidylinositol; diacylglycerol; phorbol ester; cardiac muscle; G protein-coupled receptors; phospholipase C; cell volume PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP 2 ) is the phospholipid precursor of three second messengers, D-myo-inositol 1,4,5-trisphosphate (IP 3 ), diacylglycerol (DAG), and phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) (66). At the same time, PIP 2 serves other cellular functions. It anchors and modulates the function of numerous cell signaling proteins and cytoskeleton at the cell membrane (11,17,42,65), including at least one transcription factor that is released by phospholipase C (PLC) activation (60). In addition, PIP 2 metabolism is coupled to membrane trafficking, including some forms of exo-and endocytosis (7, 46). Finally, PIP 2 modulates the function of phospholipases (14), receptor kinases (16, 52), and ion transporters and ion channels (25). Especially, the anchoring/recruitment functions and the modulatory functions of PIP 2 beg the question as to how, and if, PIP 2 might be used as a cell signal. For cardiac physiology, an answer to this question seems especially important at this time, because sarcolemmal mechanisms that affect both cardiac contraction (e.g., Na ϩ /Ca 2ϩ exchange) and contraction frequency [e.g., G protein-coupled inwardly rectifying K ϩ (GIRK) channels] are strongly PIP 2 dependent (27).The minimum biochemical mechanisms involved in cardiac myocyte PIP 2 metabolism (39) are summarized in Fig. 1. The dominant pathway of PIP 2 synthesis, as in other cells, is probably the sequential phosphorylation in the sarcolemma of phosphatidylinositol (PI) at the 4-and then the 5-positions of inositol (66). As in other cells, PIP 2 is hydr...

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum-and glucocorticoidinduced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA. WNKs5 (with no lysine (K)) are large protein-serine/threonine kinases found in all multicellular and a few unicellular eukaryotes (1). WNK1, the first member of the family identified in mammals, was found in searches for novel components of protein kinase cascades (2). WNK1 is expressed ubiquitously, consistent with effects on many cell types (2-4). Numerous splice forms, containing from just under 2000 to almost 2400 residues, are known, including one lacking most of the kinase domain (KS-WNK1), which is enriched in kidney (5, 6).The four WNK family members are distinct from all other protein kinases in that their catalytic lysine is shifted from its usual position buried in the N-terminal part of the kinase core to a more exposed position in the glycine-rich loop (2, 7). The strict conservation of the unique catalytic core structure of the WNK family in organisms such as Chlamydomonas, Phycomyces, Arabidopsis, and mammals suggests conserved properties for these kinases.Our initial characterization of WNK1 revealed that the kinase activity is sensitive to hypertonic stress (2). The subsequent discovery that WNK1 and WNK4 are genetically linked to a rare type of hypertension, pseudohypoaldosteronism type 2 (PHA2) (4), demonstrated the importance of WNK function in man and an implicit significance of the sensitivity of WNK1 kinase activity to osmotic stress. Further characterization showed that activity is increased in response to increased and decreased ionic strength (8). The consequences of WNK mutations in PHA2 are hyperkalemia, renal tubular acidosis, and eventually hypertension (1, 9 -11). WNK1 knock-out mice do not survive, but heterozygotes have low blood pressure (12), ...

The mechanistic target of rapamycin complex 1 (mTORC1) coordinates cell growth with its nutritional, hormonal, energy, and stress status. Amino acids are critical regulators of mTORC1 that permit other inputs to mTORC1 activity. However, the roles of individual amino acids and their interactions in mTORC1 activation are not well understood. Here we demonstrate that activation of mTORC1 by amino acids includes two discrete and separable steps: priming and activation. Sensitizing mTORC1 activation by priming amino acids is a prerequisite for subsequent stimulation of mTORC1 by activating amino acids. Priming is achieved by a group of amino acids that includes L-asparagine, L-glutamine, L-threonine, L-arginine, L-glycine, L-proline, L-serine, L-alanine, and L-glutamic acid. The group of activating amino acids is dominated by L-leucine but also includes L-methionine, L-isoleucine, and L-valine. L-Cysteine predominantly inhibits priming but not the activating step. Priming and activating steps differ in their requirements for amino acid concentration and duration of treatment. Priming and activating amino acids use mechanisms that are distinct both from each other and from growth factor signaling. Neither step requires intact tuberous sclerosis complex of proteins to activate mTORC1. Concerted action of priming and activating amino acids is required to localize mTORC1 to lysosomes and achieve its activation.