The Polycystic Kidney Disease 1 Gene Product Mediates Protein Kinase C α-dependent and c-Jun N-terminal Kinase-dependent Activation of the Transcription Factor AP-1*

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disorder that accounts for 8–10% of end stage renal disease. PKD1, one of two recently isolated ADPKD gene products, has been implicated in cell-cell and cell-matrix interactions. However, the signaling pathway of PKD1 remains undefined. We found that the C-terminal 226 amino acids of PKD1 transactivate an AP-1 promoter construct in human embryonic kidney cells (293T). PKD1-induced transcription is specific for AP-1; promoter constructs containing cAMP response element-binding protein, c-Fos, c-Myc, or NFκB-binding sites are unaffected by PKD1. In vitro kinase assays revealed that PKD1 triggers the activation of c-Jun N-terminal kinase (JNK), but not of mitogen-activated protein kinases p38 or p44. Dominant-negative Rac-1 and Cdc42 mutations abrogated PKD1-mediated JNK and AP-1 activation, suggesting a critical role for small GTP-binding proteins in PKD1-mediated signaling. Several protein kinase C (PKC) inhibitors decreased PKD1-mediated AP-1 activation. Conversely, expression of the C-terminal domain of PKD1 increased PKC activity in 293T cells. A dominant-negative PKC α, but not a dominant-negative PKC β or δ, abrogated PKD1-mediated AP-1 activation. These findings indicate that small GTP-binding proteins and PKC α mediate PKD1-induced JNK/AP-1 activation, together comprising a signaling cascade that may regulate renal tubulogenesis.

Mutations within the PKD1 gene on human chromosome 16p13.3 account for 85% of all diagnosed cases of ADPKD. 1 PKD1 was recently cloned and found to be broadly expressed (1)(2)(3)(4)(5). The predicted PKD1 protein is a large glycoprotein with a signal peptide and multiple transmembrane domains. Its large extracellular region of approximately 2557 amino acids contains multiple protein motifs that are associated with cellcell and cell-matrix interactions, such as leucine-rich repeats, a C-type lectin domain, immunoglobulin-like repeats, and four type III fibronectin-related domains (reviewed in Ref. 6). The short C-terminal cytoplasmic tail consists of a novel domain of 226 amino acids.
In normal human adult kidneys, cellular proliferation is a rare and unusual event. The bulk of renal mitogenesis and morphogenesis occurs before birth, with the remainder completed within the first years of life. In early development, PKD1 appears to regulate the proliferation and maturation of tubular epithelial cells. PKD1 is highly expressed in fetal kidney in areas of rapid proliferation and developmental induction, such as the ureteric bud and the condensating mesenchyme (5,7). In renal cysts of ADPKD patients, absence of PKD1 is associated with the increased expression of proto-oncogenes and growth factor receptors such as c-fos, c-myc, c-K-ras, and c-erb B2 (8 -12). Similar abnormalities have been observed in several animal models of polycystic kidney disease (13). These studies have established a critical role for PKD1 in signaling pathways controlling proto-oncogene expression, cellular proliferation, and differentiation of tubular epithelial cells.
Insight into the function of PKD1 has been hampered by its complexity. Full-length PKD1 has not yet been expressed in vivo (14), curtailing genetic manipulation of the intact protein.
Recently, we have shown heterodimeric interaction between PKD1 and PKD2 fusion proteins (15), using a heterologous integral membrane protein fused to the C-terminal cytoplasmic domain of PKD1 and PKD2. This approach has been used to delineate the effector mechanisms of various cytoplasmic domains (16 -21). In the present study, we demonstrate that the C-terminal cytoplasmic domain of PKD1 triggers the activation of AP-1, a transcription factor that modulates a variety of cellular programs, including growth response and apoptosis (reviewed in Ref. 22). Furthermore, PKD1-induced AP-1 activity was mediated at least in part through activation of protein kinase C and c-Jun N-terminal kinase (JNK). Thus, we have shown that PKD1, a protein essential for normal tubulogenesis, activates a signaling cascade involved in cellular proliferation, differentiation, and apoptosis.

EXPERIMENTAL PROCEDURES
Reagents and Plasmids-Genistein (Calbiochem), staurosporine (Calbiochem), calphostin C (Calbiochem), BAPTA-acetoxymethyl ester (Molecular Probes), wortmannin (Calbiochem), and human epidermal growth factor (Clonetics) were used at concentrations as indicated. The C-terminal domain of PKD1 was expressed as a CD16-CD7 fusion protein (CD16.7.PKD1) (15). The control vector (CD16.7) encodes the extracellular domain of CD16 followed by the CD7 transmembrane domain and a short cytoplasmic tail. The AP-1 luciferase reporter construct of four AP-1-binding sites (kindly provided by D. Moore), the collagenase promoter reporter construct containing a single AP-1-binding site at Ϫ73/63 (kindly provided by N. H. Colburn), the Jun2TRE construct of three 12-O-tetradecanoylphorbol-13-acetate-response elements (TRE) from the c-Jun promoter second TRE (kindly provided by S. Lewis and S. Hyman), the NFB construct of eight NFB-binding sites (kindly provided by B. Seed), and the c-myc construct of four c-myc-binding sites (derived from a construct kindly provided by R. N. Eisenman) all contain DNA-binding sites followed by either a minimal thymidine kinase or Rous sarcoma virus promoter directing the expression of luciferase. The c-Fos promoter construct (kindly provided by Bender GmBH) and the cAMP-response element promoter construct (kindly provided by K. Mayo) contain promoter regions of c-Fos and ␣-inhibin, respectively. The hemagglutinin (HA)-tagged p38 and p44 were kindly provided by J. Pouysségur, HA-JNK1 was kindly provided by M. Karin, the dominant-negative mutants of Rac-1 (Rac-1(N17)) and Cdc42 (Cdc42(N17)) were kindly provided by J. S. Gutkind, a dominantnegative form of PKC ␣ was kindly provided by D. Rosson, and a dominant-negative form of PKC ␤II m217 was kindly provided by C. E. Chalfant. The dominant-negative form of PKC ␦ was generated through site-directed mutagenesis replacing the lysine at 377 with arginine (23).
Luciferase Assay-293T cells seeded in 6-well plates were transiently transfected with a luciferase reporter construct, a ␤-galactosidase expression vector (kindly provided by C. Cepko), and a vector directing the expression of CD16.7.PKD1. Total DNA amount was 1-2.5 g/well. Cells were serum starved for 24 h, harvested in cold phosphate-buffered saline, and lysed in 200 l of reporter lysis buffer (Promega) for 15 min at room temperature. Lysates were centrifuged at 14,000 rpm for 3 min to remove insoluble material. Luciferase activity was determined using a commercial assay system (Promega) following the manufacturer's instructions and normalized for ␤-galactosidase activity to correct for the transfection efficiency. Pharmacological inhibitors were added for 8 h before the assay.
In Vitro Kinase Assays-The immune complex kinase assays were carried out after co-transfecting 293T cells with HA-tagged MAP kinases and the CD16.7.PKD1 construct at a 1:1 ratio. Cells from one 10-cm dish were lysed 24 h after transfection in 1 ml of cold lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , and a protease inhibitor mixture (Boehringer Mannheim). After centrifugation for 15 min at 4°C, the HA-tagged kinases were immunoprecipitated from the cleared lysate with 5 g of monoclonal anti-HA antibody (Boehringer Mannheim) for 2 h at 4°C. Immune complexes were immobilized by adding 30 l of Gamma-Bind Sepharose (Pharmacia Biotech Inc.). The immune complexes were washed three times in lysis buffer and twice in kinase reaction buffer (25 mM Hepes, 20 mM MgCl 2 , 2 mM dithiotreitol, 0.1 mM Na 3 VO 4 , pH 7.6). The immunoprecipitates were resuspended in 30 l of kinase reaction buffer containing 3 g of substrates phosphorylated heat-and acid-stable protein-1 (Stratagene) for HA-p38 and HA-p44 or GST-c-Jun (1-79) (Stratagene) for HA-JNK1. The assay was carried out in the presence of 20 M unlabeled ATP and 10 Ci of [␥-32 P]ATP for 30 min at 30°C, stopped by the addition of 30 l of SDS sample buffer, and boiled for 5 min. The reaction mixture was fractionated on a 12% SDS-PAGE. Phosphorylated substrates were visualized by autoradiography and quantitated by scanning with a Molecular Dynamics densitometer. JNK immunoprecipitates were analyzed by SDS-PAGE and Western blot, using the anti-HA monoclonal antibody in combination with a goat anti-mouse horseradish peroxidase antibody (Dako) and enhanced chemiluminescence (Pierce).
PKC Activity-PKC activities were determined using a colorimetric PKC assay with neurogranin as a dye-labeled synthetic peptide substrate (Pierce). Cells were harvested and lysed on ice for 10 min in 45 l of cold hypotonic buffer (1 mM Hepes, 5 mM MgCl 2 , 25 g/ml leupeptin, 25 g/ml pepstatin). Isotonicity was reestablished by adding 5 l of Hepes (200 mM, pH 7.4) and 25 l of an equilibrium buffer (20 mM Hepes, 5 mM MgCl 2 , 1 mM NaF, 0.1 mM Na 3 VO 4 ). The PKC reaction was performed at 30°C for 30 min using 10 l of cleared lysates following the instructions of the manufacturer. The absorbance of the phosphorylated substrates was spectrophotometrically determined at 570 nm, and a microprotein assay (Bio-Rad) was used to normalize the PKC activities for the protein content.
Western Blot Analysis-293T cells were transiently transfected by the calcium phosphate method. After incubation for 24 h, cells were lysed in sample buffer, fractionated on SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (NEN Life Science Products).
Western blot analysis was performed with an anti-CD16 monoclonal antibody followed by incubation with horseradish peroxidase-coupled goat anti-mouse immunoglobulin (Dako). Immobilized antibodies were detected by chemiluminescence (Pierce).
Statistical Analysis-Results were expressed as means Ϯ S.D. Analysis of variance with a subsequent Scheffe's test was used to determine significant difference in multiple comparisons. Values of p Ͻ 0.05 were considered to be significant.

RESULTS AND DISCUSSION
Previous studies indicate that ADPKD epithelial cells arrest in a less than terminally differentiated state, leading to cystogenesis. To identify potential signaling pathways of PKD1, we coexpressed the cytoplasmic tail of PKD1 with luciferase reporter constructs transactivated by defined DNA-binding sites. The C-terminal 226 amino acids of PKD1 were fused to the extracellular domain of CD16 and the transmembrane domain of CD7. The combination of CD16 together with a CD7 transmembrane domain has previously been shown to target cytoplasmic domains to the plasma membrane without altering effector function (18 -21) or protein-protein interaction, such as between PKD1 and PKD2 (15). The expression of CD16.7.PKD1, monitored by Western blot analysis, consistently triggered a 5-10-fold activation of the AP-1 reporter construct containing four AP-1-binding sites (Figs. 1, 3, and 5), and a more than 5-fold activation of a collagenase promoter construct containing a single AP-1 site (24) (Fig. 1). PKD1mediated AP-1 activation was highly specific, involving only AP-1 and the Jun2TRE (Fig. 1). Cytoplasmically expressed FLAG-tagged PKD1, lacking both leader sequence and transmembrane domain, completely lost the ability to activate AP-1 (data not shown), suggesting that membrane localization of PKD1 is requisite for AP-1 activation. Likewise, a control protein of the extracellular domain of CD16 fused to the transmembrane domain of CD7 had no effect (Fig. 1). AP-1 activation induced by PKD1 exceeded activation by both epidermal growth factor and serum by 2-fold (data not shown). AP-1 is a transcriptional activator composed of Jun, Fos, or activating transcription factor 2 homodimers and heterodimers that bind to a common DNA sequence, the TRE. AP-1 is activated by a large variety of extracellular stimuli, including growth hormones and cytokines; its activity is controlled both at the transcriptional level and through post-translational modifications of c-Fos and c-Jun (reviewed in Ref. 22). Several kinase cascades have been demonstrated to regulate AP-1 activity (25), including the Hog1p homolog p38, the mitogen-activated kinases p42 and p44, and members of the JNK family (reviewed in Ref. 26).
To further delineate the signaling pathway through which PKD1 may generate AP-1, we examined the activity of several kinase cascades in 293T cells expressing the C terminus of PKD1. HA-tagged p38, p44, and JNK1 were co-expressed with the C-terminal domain of PKD1 fused to CD16.7 (CD16.7.PKD1) or the control vector (CD16.7). After serum starvation for 16 h, HA-tagged kinases were immunoprecipitated, and the activity of the different kinases was determined using an in vitro kinase assay. Expression of the cytoplasmic domain of PKD1 increased JNK activity, but not that of p38 or p44 (Fig. 2). Activation of JNK was specific for PKD1 and was not detectable in 293T cells expressing full-length PKD2 (data not shown). These results together with our findings that PKD1 triggers the Jun2TRE, but not the c-Fos promoter, suggests that PKD1-induced AP-1 activation may be mediated through the formation of c-Jun and activating transcription factor 2 dimers. JNKs are typically activated by growth factors, cytokines, osmotic stress, or UV light, triggering c-Jun dimerization through phosphorylation at residues Ser-63 and Ser-73 (27,28). Many of these conditions result in the concomitant activation of p38 and JNK, although selective activation of JNK can occur. MEK kinases, specific mixed lineage kinases, transforming growth factor-␤ activated kinase, tumor progression locus 2 (Tpl-2), and p21-activated kinases have all been demonstrated to activate the JNK pathway (reviewed in Ref. 29), whereas selective activation of JNK has been reported through the specific activation of MKK4 by germinal center kinase (30). It appears that protein-activated kinases, MEK kinases, mixed lineage kinase 3, and Raf are regulated upstream by low molecular weight GTP-binding proteins (25,(31)(32)(33)(34)(35). Recently, two members of this family, Rac-1 and Cdc42, have been demonstrated to activate JNK (33).
To determine the role of small GTP-binding proteins Rac-1 and Cdc42 in the PKD1-mediated activation of JNK, we coexpressed the C-terminal domain of PKD1 with the dominantnegative mutants Rac-1(N17) and Cdc42(N17) (33). Although wild-type Rac-1 and Cdc42 had no affect on the PKD1-mediated JNK activation (data not shown), both molecules nearly abrogated the PKD1-mediated JNK activation (Fig. 3A). A comparable inhibition of PKD1-mediated AP-1 activation by Rac-1(N17) and Cdc42(N17) was observed (Fig. 3B). Thus, Rho family members Rac-1 and Cdc42 appear to play a central role in the signaling pathway triggered by the C terminus of PKD1.
To define additional downstream components of PKD1-mediated signaling, we examined the ability of various pharmacological agents to disrupt PKD1-mediated AP-1 activation. Staurosporine, BAPTA, and calphostin C inhibited PKD1-induced AP-1 activation in a dose-dependent fashion (Fig. 4), whereas genistein and wortmannin had no effect (data not shown). Consistent with the profound effect of staurosporine and calphostin C, PKD1 caused an elevation of total protein kinase C activity; a comparable increase in PKC activity was obtained after stimulation of 293T cells with phorbol 12-myristate 13-acetate (1 M) for 60 min (Fig. 5A). Because PKD1mediated PKC activation was inhibited by both staurosporine (200 nM) and BAPTA (10 M), we speculated that PKD1-induced AP-1 activation is mediated by a calcium-dependent PKC isoenzyme. PKC is a family of proteins with at least 13 different members (36), but only the conventional PKCs, ␣, ␤I, ␤II, and ␥, are regulated by calcium. A dominant-negative form of the calcium-dependent PKC ␣ (37) suppressed PKD1-mediated AP-1 activation, whereas equally well expressed dominant-negative forms of PKC ␤II and PKC ␦ had no effect (Fig.  5B). These findings suggest that PKC ␣ may mediate PKD1 signaling. PKC ␣ has been reported to activate MAP kinase and TRE/AP-1 in other signaling pathways (38 -40), perhaps by dephosphorylating c-Jun at one or more of three critical serine/ threonine residues that negatively regulate its DNA binding activity (41).
This study demonstrates that the cytoplasmic domain of PKD1 activates AP-1 and JNK and that PKC ␣, Cdc42, and Rac-1 are critical components of these signaling events. The mediators immediately downstream of PKD1 that engage these proteins are yet to be delineated. Recent reports indicate that the coiled-coil structure in the C-terminal domain of PKD1 serves as a docking site for several proteins including PKD2 (15,(42)(43)(44). In addition to several potential PKCϪ/PKAϪ and tyrosine phosphorylation sites, a putative 14-3-3-binding domain and a putative SH3-binding domain in the C terminus of PKD1 may serve as docking sites for cytoplasmic signaling proteins. Binding of 14-3-3 to the C terminus of PKD1 has recently been demonstrated in the yeast two-hybrid system (45); interestingly, 14-3-3 adapter proteins have been shown to activate certain PKC isoenzymes (46,47). Further mapping of the C terminus of PKD1, such as point mutations that destroy the 14-3-3 and SH3-binding domain, could distinguish discrete sites involved in PKD1-mediated AP-1 activation and facilitate the identification of pertinent docking proteins that modulate PKC, GTP-binding proteins, and other effectors. A role of Rho family GTP-binding proteins is to regulate the organization of the actin cytoskeleton and influence cell motility, shape, and adhesion (reviewed in Refs. 48 and 49). We postulate that aberrant activation of several small GTP-binding proteins could underlie some of the anomalies evident in ADPKD cysts, such as abnormalities of cell polarity and protein sorting (50 -55). In this model of ADPKD, disregulation of PKD1 function would pathologically affect the activation or recruitment of these small GTP-binding molecules. The role of AP-1 during renal development remains to be elucidated. We hypothesize that a burst of transcriptional activity, mediated in part by PKD1, triggers cellular proliferation and differentiation, followed by a decline in proto-oncogene expression. In cells lacking PKD1, proto-oncogene levels remain pathologically elevated. Indeed, certain genes implicated in cystogenesis in ADPKD contain AP-1-binding sites. These include ␣1 (I) collagen and several matrix metalloproteinases, genes normally involved in the synthesis and remodeling of the extracellular matrix during normal tubulogenesis (56 -60). Aberrant expression of these gene products may underlie the accumulation of an abnormal extracellular matrix in ADPKD (reviewed in Ref. 61). Another potential candidate of AP-1 regulation is hepatic nuclear factor (HNF). HNF-1 and HNF-4 are reduced in both cpk mouse and cy rat models of cystic disease (reviewed in Ref. 62), indicating a certain minimal requirement for these AP-1dependent transcription factors during renal development. Lastly, AP-1 has been implicated in the induction of differentiation (63,64) as well as apoptosis (reviewed in Ref. 22); the latter appears to be tightly controlled by Bcl-2 (65). Surprisingly, mice lacking Bcl-2 develop cystic disease despite massive apoptosis, perhaps as the result of an inbalance between Bcl-2 and PKD1 activity. Our results suggest that the C-terminal domain of PKD1 triggers cellular functions important for normal renal development. Future experiments will be necessary to analyze the PKD1 motifs and the nature of PKD1-binding adapter proteins involved in this activation. Results were compared with the PKC activity of cells stimulated with phorbol 12-myristate 13-acetate (1 M for 60 min) or untransfected cells. Results were expressed as fold increase of the vector control. The number of tests performed is indicated above the bars. Differences were highly significant (***, p Ͻ 0.001). B, a dominant-negative PKC ␣ mutant (DNM), but not a dominant-negative PKC ␤II or ␦ mutant, blocks the PKD1-mediated AP-1 activation. 293T cells were transiently co-transfected with the C-terminal domain of PKD1 (CD16.7.PKD1) or a vector control (CD16.7) and dominant-negative mutants of PKC ␣, PKC ␤, and PKC ␦ isoenzymes or PKC ␣ wild type (WT) as indicated. Transactivation of AP-1 was determined after 36 h of incubation and expressed as relative light units (RLU) after normalization for ␤-galactosidase activity. Results are composed of two different experimental sets, each representative of two experiments performed in triplicate. (***, p Ͻ 0.001; ###, significantly different from PKD1-transfected cells with p Ͻ 0.001; N.S., not significant).