SCUBE3 (Signal Peptide-CUB-EGF Domain-containing Protein 3) Modulates Fibroblast Growth Factor Signaling during Fast Muscle Development

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.     

Structural Basis for the Interaction between the Potato Virus X Resistance Protein (Rx) and Its Cofactor Ran GTPase-activating Protein 2 (RanGAP2)

The potato (Solanum tuberosum) disease resistance protein Rx has a modular arrangement that contains coiled-coil (CC), nucleotide-binding (NB), and leucine-rich repeat (LRR) domains and mediates resistance to potato virus X. The Rx N-terminal CC domain undergoes an intramolecular interaction with the Rx NB-LRR region and an intermolecular interaction with the Rx cofactor RanGAP2 (Ran GTPase-activating protein 2). Here, we report the crystal structure of the Rx CC domain in complex with the Trp-Pro-Pro (WPP) domain of RanGAP2. The structure reveals that the Rx CC domain forms a heterodimer with RanGAP2, in striking contrast to the homodimeric structure of the CC domain of the barley disease resistance protein MLA10. Structure-based mutagenesis identified residues from both the Rx CC domain and the RanGAP2 WPP domain that are crucial for their interaction and function in vitro and in vivo. Our results reveal the molecular mechanism underlying the interaction of Rx with RanGAP2 and identify the distinct surfaces of the Rx CC domain that are involved in intramolecular and intermolecular interactions.     

Structure of the Type VI Effector-Immunity Complex (Tae4-Tai4) Provides Novel Insights into the Inhibition Mechanism of the Effector by Its Immunity Protein

The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently been found to play a role in interspecies interactions. The Gram-negative bacteria harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor cells against the toxic effectors. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity pairs. Here, we report the crystal structures of Tae4 from Enterobacter cloacae and Tae4-Tai4 complexes from both E. cloacae and Salmonella typhimurium. Tae4 acts as a dl-endopeptidase and displays a typical N1pC/P60 domain. Unlike Tsi1 (type VI secretion immunity 1), Tai4 is an all-helical protein and forms a dimer in solution. The small angle x-ray scattering study combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution. Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix (α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4 activity. The inhibition process requires collaboration between the Tai4 dimer. These results reveal a novel and unique inhibition mechanism in effector-immunity pairs and suggest a new strategy to develop antipathogen drugs.     

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation.     

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.     

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.     

The L279P Mutation of Nuclear Distribution Gene C (NudC) Influences Its Chaperone Activity and Lissencephaly Protein 1 (LIS1) Stability

LIS1, a gene mutated in classical lissencephaly, plays essential roles in cytoplasmic dynein regulation, mitosis and cell migration. However, the regulation of LIS1 (lissencephaly protein 1) protein remains largely unknown. Genetic studies in Aspergillus nidulans have uncovered that the Nud (nuclear distribution) pathway is involved in the regulation of cytoplasmic dynein complex and a temperature-sensitive mutation in the nudC gene (L146P) greatly reduces the protein levels of NudF, an Aspergillus ortholog of LIS1. Here, we showed that L146 in Aspergillus NudC and its flanking region were highly conservative during evolution. The similar mutation in human NudC (L279P) obviously led to reduced LIS1 and cellular phenotypes similar to those of LIS1 down-regulation. To explore the underlying mechanism, we found that the p23 domain-containing protein NudC bound to the molecular chaperone Hsp90, which is also associated with LIS1. Inhibition of Hsp90 chaperone function by either geldanamycin or radicicol resulted in a decrease in LIS1 levels. Ectopic expression of Hsp90 partially reversed the degradation of LIS1 caused by overexpression of NudC-L279P. Furthermore, NudC was found to regulate the ATPase activity of Hsp90, which was repressed by the mutation of L279P. Interestingly, NudC itself was shown to possess a chaperone function, which also was suppressed by the L279P mutation. Together, these data suggest that NudC may be involved in the regulation of LIS1 stability by its chaperone function.     

A Small GTPase Activator Protein Interacts with Cytoplasmic Phytochromes in Regulating Root Development

Phytochromes enable plants to sense light information and regulate developmental responses. Phytochromes interact with partner proteins to transmit light signals to downstream components for plant development. PIRF1 (phytochrome-interacting ROP guanine-nucleotide exchange factor (RopGEF 1)) functions as a light-signaling switch regulating root development through the activation of ROPs (Rho-like GTPase of plant) in the cytoplasm. In vitro pulldown and yeast two-hybrid assays confirmed the interaction between PIRF1 and phytochromes. PIRF1 interacted with the N-terminal domain of phytochromes through its conserved PRONE (plant-specific ROP nucleotide exchanger) region. PIRF1 also interacted with ROPs and activated them in a phytochrome-dependent manner. The Pr form of phytochrome A enhanced the RopGEF activity of PIRF1, whereas the Pfr form inhibited it. A bimolecular fluorescence complementation analysis demonstrated that PIRF1 was localized in the cytoplasm and bound to the phytochromes in darkness but not in light. PIRF1 loss of function mutants (pirf1) of Arabidopsis thaliana showed a longer root phenotype in the dark. In addition, both PIRF1 overexpression mutants (PIRF1-OX) and phytochrome-null mutants (phyA-211 and phyB-9) showed retarded root elongation and irregular root hair formation, suggesting that PIRF1 is a negative regulator of phytochrome-mediated primary root development. We propose that phytochrome and ROP signaling are interconnected through PIRF1 in regulating the root growth and development in Arabidopsis.     

Cloning and Characterization of AtRGP1 : A Reversibly Autoglycosylated Arabidopsis Protein Implicated in Cell Wall Biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.The primary cell wall of dicot plants is laid down by young cells prior to the cessation of elongation and secondary wall deposition. Making up to 90% of the cell''s dry weight, the extracellular matrix is important for many processes, including morphogenesis, growth, disease resistance, recognition, signaling, digestibility, nutrition, and decay. The composition of the cell wall has been extensively described (Bacic et al., 1988; Levy and Staehelin, 1992; Zablackis et al., 1995), and yet many questions remain unanswered regarding the synthesis and interaction of these components to provide cells with a functional wall (Carpita and Gibeaut, 1993; Carpita et al., 1996).Heteropolysaccharide biosynthesis can be divided into four steps: (a) chain or backbone initiation, (b) elongation, (c) side-chain addition, and (d) termination and extracellular deposition (Waldron and Brett, 1985). The similarity between various polysaccharide backbones leads to the prediction that the synthesizing machinery would be conserved between them. For example, the backbone of xyloglucan polymers, β-1,4 glucan, can be synthesized independently of or concurrently with side-chain addition (Campbell et al., 1988; White et al., 1993), and this polymer and the chains that make up cellulose are identical. The later addition of side chains to xyloglucan are catalyzed by specific transferases (Kleene and Berger, 1993) such as xylosyltransferase (Campbell et al., 1988), galactosyltransferase, and fucosyltransferase (Faïk et al., 1997), all of which are localized to the Golgi compartment (Brummell et al., 1990; Driouich et al., 1993; Staehelin and Moore, 1995).The enzymes involved in wall biosynthesis have been recalcitrant to isolation (Carpita et al., 1996; Albersheim et al., 1997). Only recently has the first gene encoding putative cellulose biosynthetic enzymes, celA, been isolated from cotton (Gossypium hirsutum) and rice (Oryza sativa; Pear et al., 1996).During studies of polysaccharide synthesis in pea (Pisum sativum) Golgi membranes, Dhugga et al. (1991) identified a 41-kD protein doublet that they suggested was involved in polysaccharide synthesis. The authors showed that this protein could be glycosylated by radiolabeled UDP-Glc but that this labeling could be reversibly competed with by unlabeled UDP-Glc, UDP-Xyl, and UDP-Gal, the sugars that make up xyloglucan (Hayashi, 1989). The 41-kD protein was named PsRGP1 (P. sativum Reversibly Glycosylated Polypeptide-1; Dhugga et al., 1997). Furthermore, the conditions that stimulate or inhibit Golgi-localized β-glucan synthase activity are the same conditions that stimulate or inhibit the glycosylation of PsRGP1 (Dhugga et al., 1991). To address the role of this protein in polysaccharide synthesis, the authors purified the polypeptides and obtained the sequences from tryptic peptides (Dhugga and Ray, 1994). Antibodies raised against PsRGP1 showed that it is soluble and localized to the plasma membrane (Dhugga et al., 1991) and Golgi compartment (Dhugga et al., 1997). In addition to its Golgi localization, the steady-state glycosylation of PsRGP1 is approximately 10:7:3 (UDP-Glc:-Xyl:-Gal), which is similar to the typical sugar composition of xyloglucan (1.0:0.75:0.25; Dhugga et al., 1997).We were interested in studying various aspects of cell wall metabolism, including the synthesis of polysaccharides and their delivery to the cell wall. Studies in pea have shown that a 41-kD protein may be involved in cell wall polysaccharide synthesis, possibly that of xyloglucan (Dhugga et al., 1997). Here we report the characterization of AtRGP1 (Arabidopsis thaliana Reversibly Glycosylated Polypeptide-1), a soluble protein that can also be found weakly associated with membrane fractions, most likely the Golgi fraction. The reversible nature of the glycosylation of this Arabidopsis homolog by the substrates used to make polysaccharides (nucleotide sugars) suggests a possible role for AtRGP1 in polysaccharide biosynthesis.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

TopBP1 (topoisomerase IIβ-binding protein 1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA replication has been partially elucidated; however, its role in the checkpoint response remained elusive. In this study, we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin at Ser-1000 is important for its checkpoint activity. Overall, our results indicate that, like TopBP1, Treslin is a dual replication/checkpoint protein that directly participates in ATR-mediated checkpoint signaling.     

Amer2 Protein Interacts with EB1 Protein and Adenomatous Polyposis Coli (APC) and Controls Microtubule Stability and Cell Migration

EB1 is key factor in the organization of the microtubule cytoskeleton by binding to the plus-ends of microtubules and serving as a platform for a number of interacting proteins (termed +TIPs) that control microtubule dynamics. Together with its direct binding partner adenomatous polyposis coli (APC), EB1 can stabilize microtubules. Here, we show that Amer2 (APC membrane recruitment 2), a previously identified membrane-associated APC-binding protein, is a direct interaction partner of EB1 and acts as regulator of microtubule stability together with EB1. Amer2 binds to EB1 via specific (S/T)xIP motifs and recruits it to the plasma membrane. Coexpression of Amer2 and EB1 generates stabilized microtubules at the plasma membrane, whereas knockdown of Amer2 leads to destabilization of microtubules. Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated down-regulation of Xenopus Amer2 blocks convergent extension cell movements, suggesting that the Amer2-EB1-APC complex regulates cell migration by altering microtubule stability.     

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.     

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish,Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities.     

Lipid Droplet Protein LID-1 Mediates ATGL-1-Dependent Lipolysis during Fasting in Caenorhabditis elegans

Lipolysis is a delicate process involving complex signaling cascades and sequential enzymatic activations. In Caenorhabditis elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, we demonstrate that in the intestinal cells of C. elegans, a newly identified protein, lipid droplet protein 1 (C25A1.12; LID-1), modulates lipolysis by binding to adipose triglyceride lipase 1 (C05D11.7; ATGL-1) during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNA interference (RNAi) resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. In adult worms, short-term fasting increased cyclic AMP (cAMP) levels, which activated protein kinase A (PKA) to stimulate lipolysis via ATGL-1 and LID-1. Moreover, ATGL-1 protein stability and LID-1 binding were augmented by PKA activation, eventually leading to increased lipolysis. These data suggest the importance of the concerted action of lipase and lipid droplet protein in the response to fasting signals via PKA to maintain lipid homeostasis.     

WNK4 Kinase Stimulates Caveola-mediated Endocytosis of TRPV5 Amplifying the Dynamic Range of Regulation of the Channel by Protein Kinase C

WNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca²⁺ channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca²⁺ reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (∼215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca²⁺ reabsorption by parathyroid hormones or the tissue kallikrein in vivo.     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.