A high-resolution structure of the EF-hand domain of human polycystin-2

Autosomal dominant polycystic kidney disease (ADPKD) affects over 1:1000 of the worldwide population and is caused by mutations in two genes, PKD1 and PKD2. PKD2 encodes a 968-amino acid membrane spanning protein, Polycystin-2 (PC-2), which is a member of the TRP ion channel family. The C-terminal cytoplasmic tail contains an EF-hand motif followed by a short coiled-coil domain. We have determined the structure of the EF-hand region of PC-2 using NMR spectroscopy. The use of different boundaries, compared with those used in previous studies, have enabled us to determine a high resolution structure and show that the EF hand motif forms a standard calcium-binding pocket. The affinity of this pocket for calcium has been measured and mutants that both decrease and increase its affinity for the metal ion have been created.     

A short carboxy-terminal domain of polycystin-1 reorganizes the microtubular network and the endoplasmic reticulum

Mutations of PKD1 cause autosomal dominant polycystic kidney disease (ADPKD), a syndrome characterized by kidney cysts and progressive renal failure. Polycystin-1, the protein encoded by PKD1, is a large integral membrane protein with a short carboxy-terminal cytoplasmic domain that appears to initiate multiple cellular programs. We report now that this polycystin-1 domain contains a novel motif responsible for rearrangements of intermediate filaments, microtubules and the endoplasmic reticulum (ER). This motif reveals homology to CLIMP-63, a microtubule-binding protein that rearranges the ER. Our findings suggest that polycystin-1 influences the shape and localization of both the microtubular network and the ER.     

Carboxy Terminal Tail of Polycystin-1 Regulates Localization of TSC2 to Repress mTOR

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited renal disorder caused by defects in the PKD1 or PKD2 genes. ADPKD is associated with significant morbidity, and is a major underlying cause of end-stage renal failure (ESRF). Commonly, treatment options are limited to the management of hypertension, cardiovascular risk factors, dialysis, and transplantation when ESRF develops, although several new pharmacotherapies, including rapamycin, have shown early promise in animal and human studies. Evidence implicates polycystin-1 (PC-1), the gene product of the PKD1 gene, in regulation of the mTOR pathway. Here we demonstrate a mechanism by which the intracellular, carboxy-terminal tail of polycystin-1 (CP1) regulates mTOR signaling by altering the subcellular localization of the tuberous sclerosis complex 2 (TSC2) tumor suppressor, a gatekeeper for mTOR activity. Phosphorylation of TSC2 at S939 by AKT causes partitioning of TSC2 away from the membrane, its GAP target Rheb, and its activating partner TSC1 to the cytosol via 14-3-3 protein binding. We found that TSC2 and a C-terminal polycystin-1 peptide (CP1) directly interact and that a membrane-tethered CP1 protects TSC2 from AKT phosphorylation at S939, retaining TSC2 at the membrane to inhibit the mTOR pathway. CP1 decreased binding of 14-3-3 proteins to TSC2 and increased the interaction between TSC2 and its activating partner TSC1. Interestingly, while membrane tethering of CP1 was required to activate TSC2 and repress mTOR, the ability of CP1 to inhibit mTOR signaling did not require primary cilia and was independent of AMPK activation. These data identify a unique mechanism for modulation of TSC2 repression of mTOR signaling via membrane retention of this tumor suppressor, and identify PC-1 as a regulator of this downstream component of the PI3K signaling cascade.     

Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration.     

Polycystin-1 activates and stabilizes the polycystin-2 channel

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder largely caused by mutations in the PKD1 and PKD2 genes that encode the transmembrane proteins polycystin-1 and -2, respectively. Both proteins appear to be involved in the regulation of cell growth and maturation, but the precise mechanisms are not yet well defined. Polycystin-2 has recently been shown to function as a Ca(2+)-permeable, non-selective cation channel. Polycystin-2 interacts through its cytoplasmic carboxyl-terminal region with a coiled-coil motif in the cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of this interaction on its channel activity, however, are unknown. In this report, we show that P1CC enhanced the channel activity of polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type polycystin-2 channel activity. The modulation of polycystin-2 channel activity by polycystin-1 may be important for the various biological processes mediated by this molecular complex.     

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.     

Functional characterization of the C. elegans nephrocystins NPHP-1 and NPHP-4 and their role in cilia and male sensory behaviors

Autosomal dominant polycystic kidney disease (ADPKD) and nephronophthisis (NPH) share two common features: cystic kidneys and ciliary localized gene products. Mutation in either the PKD1 or PKD2 gene accounts for 95% of all ADPKD cases. Mutation in one of four genes (NPHP1-4) results in nephronophthisis. The NPHP1, NPHP2, PKD1, and PKD2 protein products (nephrocystin-1, nephrocystin-2 or inversin, polycystin-1, and polycystin-2, respectively) localize to primary cilia of renal epithelia. However, the relationship between the nephrocystins and polycystins, if any, is unknown. In the nematode Caenorhabditis elegans, the LOV-1 and PKD-2 polycystins localize to male-specific sensory cilia and are required for male mating behaviors. To test the hypothesis that ADPKD and NPH cysts arise from a common defect in cilia, we characterized the C. elegans homologs of NPHP1 and NPHP4. C. elegans nphp-1 and nphp-4 are expressed in a subset of sensory neurons. GFP-tagged NPHP-1 and NPHP-4 proteins localize to ciliated sensory endings of dendrites and colocalize with PKD-2 in male-specific sensory cilia. The cilia of nphp-1(ok500) and nphp-4(tm925) mutants are intact. nphp-1; nphp-4 double, but not single, mutant males are response defective. We propose that NPHP-1 and NPHP-4 proteins play important and redundant roles in facilitating ciliary sensory signal transduction.     

The C-terminal tail of the polycystin-1 protein interacts with the Na,K-ATPase alpha-subunit

Polycystin-1 (PC-1) is the product of the PKD1 gene, which is mutated in autosomal dominant polycystic kidney disease. We show that the Na,K-ATPase alpha-subunit interacts in vitro and in vivo with the final 200 amino acids of the polycystin-1 protein, which constitute its cytoplasmic C-terminal tail. Functional studies suggest that this association may play a role in the regulation of the Na,K-ATPase activity. Chinese hamster ovary cells stably expressing the entire PC-1 protein exhibit a dramatic increase in Na,K-ATPase activity, although the kinetic properties of the enzyme remain unchanged. These data indicate that polycystin-1 may contribute to the regulation of Na,K-ATPase activity in kidneys in situ, thus modulating renal tubular fluid and electrolyte transport.     

Polycystins as components of large multiprotein complexes of polycystin interactors

Naturally occurring mutations in two separate genes, PKD1 and PKD2, are responsible for the vast majority of all cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases affecting 1 in 1000 Americans. The hallmark of ADPKD is the development of epithelial cysts in the kidney, liver, and pancreas. PKD1 encodes a large plasma membrane protein (PKD1, PC1, or Polycystin-1) with a long extracellular domain and has been speculated to function as an atypical G protein coupled receptor. PKD2 encodes an ion channel of the Transient Receptor Potential superfamily (TRPP2, PKD2, PC2, or Polycystin-2). Despite the identification of these genes more than 20 years ago, the molecular function of their encoded proteins and the mechanism(s) by which mutations in PKD1 and PKD2 cause ADPKD remain elusive. Genetic, biochemical, and functional evidence suggests they form a multiprotein complex present in multiple locations in the cell, including the plasma membrane, endoplasmic reticulum, and the primary cilium. Over the years, numerous interacting proteins have been identified using directed and unbiased approaches, and shown to modulate function, cellular localization, and protein stability and turnover of Polycystins. Delineation of the molecular composition of the Polycystin complex can have a significant impact on understanding their cellular function in health and disease states and on the identification of more specific and effective therapeutic targets.     

Polycystin-1 induces cell migration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependent cell cell mechanical adhesion

Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1-/- mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and beta-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated beta-catenin through activation of GSK3beta. Expression of a nondegradable form of beta-catenin in PC-1 MDCK cells restores strong cell-cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis.     

Structure-function relationships of the extracellular domain of the autosomal dominant polycystic kidney disease-associated protein,polycystin-1

Polycystin-1 (PC-1) is a member of a novel family of proteins that have a multidomain structure. Although the C-terminal intracellular segments have been extensively studied, mainly with respect to their putative involvement in cell signalling, the potential function of the extracellular domains has received less attention. Mutations in PC-1 result in autosomal dominant polycystic kidney disease (ADPKD) which is characterised by perturbation of transport resulting in fluid accumulation, cell proliferation and modification of the extracellular matrix. The possibility that the interaction of a component of the extracellular matrix or some external factor with PC-1 may be important in the initiation or progression of ADPKD cannot currently be ruled out. The purpose of this review is to assess current evidence for the function of the PC-1 extracellular domains, and their potential implications for ADPKD.     

A Novel Mouse Model Reveals that Polycystin-1 Deficiency in Ependyma and Choroid Plexus Results in Dysfunctional Cilia and Hydrocephalus

Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (∼520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.     

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.     

Phosphorylation, protein kinases and ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by renal cyst formation and caused by mutations in the PKD1 and PKD2 genes, which encode polycystin-1(PC-1) and -2 (PC-2) proteins, respectively. PC-1 is a large plasma membrane receptor involved in the regulation of several biological functions and signaling pathways including the Wnt cascade, AP-1, PI3kinase/Akt, GSK3β, STAT6, Calcineurin/NFAT and the ERK and mTOR cascades. PC-2 is a calcium channel of the TRP family. The two proteins form a functional complex and prevent cyst formation, but the precise mechanism(s) involved remains unknown. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Quantifying intramolecular binding in multivalent interactions: a structure-based synergistic study on Grb2-Sos1 complex

Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment.     

Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions

Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.     

SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2-tail dissociation. These results identify SH3-linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.