Steve Stippec

Differential Effects of PAK1-activating Mutations Reveal Activity-dependent and -independent Effects on Cytoskeletal Regulation*

PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.

The roles of MAPKs in disease

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.

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism.

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.

Regulation of WNK1 by an autoinhibitory domain and autophosphorylation.

WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.

SLC26A9 is a Cl- channel regulated by the WNK kinases

SLC26A9 is a member of the SLC26 family of anion transporters, which is expressed at high levels in airway and gastric surface epithelial cells. The transport properties and regulation of SLC26A9, and thus its physiological function, are not known. Here we report that SLC26A9 is a highly selective Cl− channel with minimal OH−/HCO3− permeability that is regulated by the WNK kinases. Expression in Xenopus oocytes and simultaneous measurement of membrane potential or current, intracellular pH (pHi) and intracellular Cl− (Cl−i) revealed that expression of SLC26A9 resulted in a large Cl− current. SLC26A9 displays a selectivity sequence of I− > Br− > NO3− > Cl− > Glu−, but it conducts Br− > Cl− > I− > NO3− > Glu−, with NO3− and I− inhibiting the Cl− conductance. Similarly, expression of SLC26A9 in HEK cells resulted in a large Cl− current. Although detectable, OH− and HCO3− fluxes in oocytes expressing SLC26A9 were very small. Moreover, HCO3− had no discernable effect on the Cl− current, the reversal potential in the presence or absence of Cl−o and, importantly, HCO3− had no effect on Cl− fluxes. These findings indicate that SLC26A9 is a Cl− channel with minimal OH−/HCO3− permeability. Co‐expression of SLC26A9 with the WNK kinases WNK1, WNK3 or WNK4 inhibited SLC26A9 activity, and the inhibition was independent of WNK kinase activity. Immunolocalization in oocytes and cell surface biotinylation in HEK cells indicated that the WNK‐mediated inhibition of SLC26A9 activity is caused by reduced SLC26A9 surface expression. Expression of SLC26A9 in the airway and the response of the WNKs to homeostatic stress raise the possibility that SLC26A9 serves to mediate the response of the airway to stress.

WNK1 activates SGK1 by a phosphatidylinositol 3-kinase-dependent and non-catalytic mechanism

WNK1 (with no lysine (K) 1) is a protein-serine/threonine kinase with a unique catalytic site organization. Deletions in the first intron of the WNK1 gene were found in a group of hypertensive patients with pseudohypoaldosteronism type II. No changes in coding sequence of WNK1 were found, but its expression was increased severalfold. We have been investigating actions of WNK1 and have found that WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1, which impacts membrane expression of the epithelial sodium channel. Here we explore the role of WNK1 in SGK1 regulation. Activation of SGK1 by WNK1 is blocked by phosphatidylinositol 3-kinase inhibitors. Neither the catalytic activity nor the kinase domain of WNK1 is required; rather the N-terminal 220 residues of WNK1 are necessary and sufficient to activate SGK1. Phosphorylation of WNK1 on Thr-58 contributes to SGK1 activation. Finally, we show that WNK1 is required for the activation of SGK1 by insulin-like growth factor 1.

Serum and Glucocorticoid-induced Kinase (SGK) 1 and the Epithelial Sodium Channel Are Regulated by Multiple with No Lysine (WNK) Family Members

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 glucocorticoid-induced 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.

WNK1: analysis of protein kinase structure, downstream targets, and potential roles in hypertension

ABSTRACTThe WNK kinases are a recently discovered family of serine-threonine kinases that have been shown to play an essential role in the regulation of electrolyte homeostasis. Intronic deletions in the WNK1 gene result in its overexpression and lead to pseudohypoaldosteronism type II, a disease with salt-sensitive hypertension and hyperkalemia. This review focuses on the recent evidence elucidating the structure of the kinase domain of WNK1 and functions of these kinases in normal and disease physiology. Their functions have implications for understanding the biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. The WNK kinases may be able to influence ion homeostasis through its effects on synaptotagmin function.

Biological Cross-talk between WNK1 and the Transforming Growth Factor β-Smad Signaling Pathway

WNKs (with no lysine (K)), unique serine/threonine protein kinases, have been best studied in the context of cell volume regulation and ion homeostasis. Here we describe a biological link between WNKs and transforming growth factor (TGF) β-Smad signaling. Both WNK1 and WNK4 directly bind to and phosphorylate Smad2. Knockdown of WNK1 in HeLa cells using small interfering RNA reduces Smad2 protein expression; this decrease is at least partially due to down-regulation of Smad2 transcription. In contrast, phosphorylated Smad2 significantly accumulated in the nucleus as a consequence of depletion of WNK1, resulting in Smad-mediated transcriptional responses. In addition, TGFβ-induced target gene transcripts were increased in WNK1 small interfering RNA cells. These findings suggest WNK1 as a dual modulator of TGFβ-Smad signaling pathways.

Regulation of a Third Conserved Phosphorylation Site in SGK1

SGK1 (serum- and glucocorticoid-induced kinase 1) is a member of the AGC branch of the protein kinase family. Among well described functions of SGK1 is the regulation of epithelial transport through phosphorylation of the ubiquitin protein ligase Nedd4-2 (neuronal precursor cell expressed developmentally down-regulated 4-2). The activation of SGK1 has been widely accepted to be dependent on the phosphorylation of Thr256 in the activation loop and Ser422 in the hydrophobic motif near the C terminus. Here, we report the identification of two additional phosphorylation sites, Ser397 and Ser401. Both are required for maximum SGK1 activity induced by extracellular agents or by coexpression with other protein kinases, with the largest loss of activity from mutation of Ser397. Coexpression with active Akt1 increased the phosphorylation of Ser397 and thereby SGK1 kinase activity. SGK1 activation was further augmented by coexpression with the protein kinase WNK1 (with no lysine kinase 1). These findings reveal further complexity underlying the regulation of SGK1 activity.