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• W2083021738 abstract "Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane. Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane. Protein kinase D (PKD) 2The abbreviations used are: PKDprotein kinase DPKCprotein kinase CNHERFNa+/H+ exchanger regulatory factorFRETfluorescence resonance energy transferDKARD kinase activity reporterDAGdiacylglycerolGPCRG protein-coupled receptorPLCphospholipase CPDBuphorbol-12,13-dibutyrateERMezrin-radixin-moesinCTcarboxyl-terminalGSTglutathione S-transferaseCKARC kinase activity reporterCFPcyan fluorescent proteinYFPyellow fluorescent proteinMDCKMadin-Darby canine kidneysiRNAsmall interfering RNA. plays a role in numerous processes including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1.Toker A. EMBO Rep. 2005; 6: 310-314Crossref PubMed Scopus (34) Google Scholar). The PKD family consists of three members belonging to the Ca2+/calmodulin-dependent kinase group of serine/threonine protein kinases. Each isoform contains a conserved catalytic core and an amino-terminal regulatory moiety. This regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain that autoinhibits the kinase (2.Rykx A. De Kimpe L. Mikhalap S. Vantus T. Seufferlein T. Vandenheede J.R. Van Lint J. FEBS Lett. 2003; 546: 81-86Crossref PubMed Scopus (196) Google Scholar). The C1 domains are membrane-targeting modules that bind diacylglycerol (DAG) and its functional analogues, phorbol esters, thus recruiting PKD to membranes (3.Kazanietz M.G. Mol. Pharmacol. 2002; 61: 759-767Crossref PubMed Scopus (202) Google Scholar). The PKD1 and PKD2 isoforms additionally contain PDZ-binding motifs at their carboxyl termini that can target the kinases to distinct subcellular scaffolds through interactions with PDZ domain-containing proteins (4.Sánchez-Ruiloba L. Cabrera-Poch N. Rodríguez-Martínez M. López-Menéndez C. Jean-Mairet R.M. Higuero A.M. Iglesias T. J. Biol. Chem. 2006; 281: 18888-18900Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). protein kinase D protein kinase C Na+/H+ exchanger regulatory factor fluorescence resonance energy transfer D kinase activity reporter diacylglycerol G protein-coupled receptor phospholipase C phorbol-12,13-dibutyrate ezrin-radixin-moesin carboxyl-terminal glutathione S-transferase C kinase activity reporter cyan fluorescent protein yellow fluorescent protein Madin-Darby canine kidney small interfering RNA. PKD transduces signals downstream of the second messenger DAG. In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation by novel protein kinase C (PKC) family members at two sites within its catalytic core (5.Iglesias T. Waldron R.T. Rozengurt E. J. Biol. Chem. 1998; 273: 27662-27667Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6.Rozengurt E. Rey O. Waldron R.T. J. Biol. Chem. 2005; 280: 13205-13208Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The novel PKCs themselves contain C1 domains and are allosterically activated by DAG-mediated membrane binding; thus, DAG production leads to PKD activation through coincident activation of the novel PKCs and localization of PKD near its upstream kinases. Hence, activation of phospholipase C (PLC)-coupled receptors (such as certain G protein-coupled receptors (GPCRs) or receptor tyrosine kinases) results in the production of second messengers including DAG, and this leads to recruitment and activation of the novel PKCs and thus also PKD. PDZ (PSD-95, Discs large, ZO-1) domains are compact, globular structures of ∼90 residues, occurring in one or multiple copies within a protein, that mediate protein-protein interactions (7.Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). These interactions occur via binding to other PDZ domains or, more commonly, by recognition of short amino acid motifs in the carboxyl termini of target proteins commonly terminating in a hydrophobic residue (8.Stiffler M.A. Chen J.R. Grantcharova V.P. Lei Y. Fuchs D. Allen J.E. Zaslavskaia L.A. MacBeath G. Science. 2007; 317: 364-369Crossref PubMed Scopus (305) Google Scholar). In the case of PKD1 and PKD2, the last four amino acids are VSIL and ISVL, respectively. Here we identify Na+/H+ exchanger regulatory factor 1 (NHERF-1) as a PDZ domain-containing protein that interacts with the PDZ-binding motif of both PKD1 and PKD2. NHERF-1 was originally cloned as a critical protein component for the inhibition of Na+/H+ exchanger 3 by protein kinase A (9.Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar). NHERF-1 is 52% identical to NHERF-2, a family member with which it shares the conserved domain structure of two PDZ domains followed by an ezrin-radixin-moesin (ERM)-binding region (10.Donowitz M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. 2005; 567: 3-11Crossref PubMed Scopus (182) Google Scholar). Parallel studies demonstrating its ability to strongly interact with ezrin independently identified NHERF-1 as ERM-binding phosphoprotein 50 (11.Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar). Via this ERM-binding region, NHERF-1 and NHERF-2 are predominantly localized near the actin cytoskeleton, thus poising them near the plasma membrane where they function as scaffolds. Since these original cloning reports, numerous studies have identified over 30 binding partners of these scaffold proteins including GPCRs, tyrosine kinase receptors, other adaptor proteins, signaling enzymes, and ion channels (12.Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiology. 2004; 19: 362-369Crossref PubMed Scopus (135) Google Scholar, 13.Weinman E.J. Hall R.A. Friedman P.A. Liu-Chen L.Y. Shenolikar S. Annu. Rev. Physiol. 2006; 68: 491-505Crossref PubMed Scopus (156) Google Scholar). Here we identify PKD1 and PKD2 as NHERF-1-interacting proteins. Using a fluorescence resonance energy transfer (FRET)-based assay to assess molecular proximity, both PKD1 and PKD2 are shown to transiently associate with NHERF-1 following PKD activation. Furthermore, through use of genetically encoded reporters for PKD activity, we show a unique signature of PKD activation at the NHERF scaffold. Specifically, signaling is more tightly regulated at the scaffold than in the cytosol or bulk plasma membrane. Phosphatase activity is higher at NHERF than at the plasma membrane, resulting in a more rapidly reversible PKD response at the scaffold, and following an agonist-evoked response, PKD signaling is prolonged compared with the length of response in the cytosol. Our data identify NHERF-1 as a novel nexus of PKD signaling and raise the possibility that PKD may act as a novel regulator of proteins at the NHERF scaffold. Phorbol 12,13-dibutyrate (PDBu), Gö 6976, and Gö 6983 were obtained from Calbiochem. Histamine was from Sigma-Aldrich. Restriction enzymes, T4 ligase, and Taq polymerase were obtained from New England Biolabs. LR and BP clonase were obtained from Invitrogen. NHERF-1 (ERM-binding phosphoprotein 50) antibody was obtained from Abcam. Phospho-PKD substrate antibody was from Cell Signaling Technology. All other materials were reagent grade. DNA encoding the carboxyl-terminal (CT) 25 amino acids of PKD1 or PKD2 was ligated into the pGEX-6P-3 vector (Amersham Biosciences) to generate an in-frame fusion to GST. DKAR has been described previously (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). δCKAR was generated by modification of the peptide sequence in CKAR (15.Violin J.D. Zhang J. Tsien R.Y. Newton A.C. J. Cell Biol. 2003; 161: 899-909Crossref PubMed Scopus (456) Google Scholar) to one specifically phosphorylated by PKCδ. 3T. Kajimoto and A. C. Newton, unpublished data. All of the CFP and YFP DNA utilized were the monomeric versions harboring the A206K mutations (16.Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1790) Google Scholar). The membrane-targeted CFP (MyrPalmCFP) was described previously (15.Violin J.D. Zhang J. Tsien R.Y. Newton A.C. J. Cell Biol. 2003; 161: 899-909Crossref PubMed Scopus (456) Google Scholar). DNA encoding the last 10 amino acids of PHLPP2 (17.Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar) was fused in-frame to CFP, DKAR, and δCKAR to create CFP-PDZ, DKAR-PDZ, and δCKAR-PDZ, respectively. FLAG-NHERF-1 was used in overexpression studies and has been described previously (18.Lau A.G. Hall R.A. Biochemistry. 2001; 40: 8572-8580Crossref PubMed Scopus (102) Google Scholar). CFP-NHERF-1 was generated by subcloning NHERF-1 DNA into a plasmid containing monomeric CFP, resulting in an in-frame fusion to the amino terminus of NHERF-1. mCherry-NHERF-1 was generated by Gateway cloning (Invitrogen) into a Gateway destination vector for NH2-terminal fusion of monomeric Cherry (mCherry) (19.Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 7877-7882Crossref PubMed Scopus (2007) Google Scholar). YFP was ligated to PKD1 and PKD2 to create amino-terminally tagged PKD1 and PKD2 (YFP-PKD1 and YFP-PKD2, respectively). YFP-PKD2ΔPDZ was generated by deleting the last three amino acids of PKD2 within YFP-PKD2 following the QuikChange protocol (Stratagene). Expression of GST, GST-PKD1-CT, and GST-PKD2-CT was induced for 4 h with 0.3 mm isopropyl β-d-thiogalactopyranoside in BL21(DE3) Escherichia coli. Pelleted cells were homogenized in 50 mm Tris, pH 7.5, 200 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, containing 300 nm phenylmethylsulfonyl fluoride, 500 nm benzamidine, 500 ng ml−1 leupeptin, and 1 mg ml−1 lysozyme and rocked for 30 min at 4 °C followed by brief bath sonication. The lysate was treated with 100 μg ml−1 DNase and cleared by centrifugation at 14,000 × g for 30 min at 4 °C. GST fusion proteins were purified from the lysate using the Profinia Protein Purification System according to the manufacturer's specifications (Bio-Rad). The eluted, purified protein was dialyzed against 20 mm HEPES, pH 7.5, 50 mm NaCl. Membranes containing 96 putative PDZ domains were prepared as described (20.Fam S.R. Paquet M. Castleberry A.M. Oller H. Lee C.J. Traynelis S.F. Smith Y. Yun C.C. Hall R.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8042-8047Crossref PubMed Scopus (82) Google Scholar). 1 mg ml−1 of purified GST, GST-PKD1-CT, or GST-PKD2-CT fusion protein was overlaid onto the array and detected by far Western blotting as described previously (18.Lau A.G. Hall R.A. Biochemistry. 2001; 40: 8572-8580Crossref PubMed Scopus (102) Google Scholar). MDCK cells were maintained in Dulbecco's modified Eagle's medium/F-12 (Cellgro) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Cellgro) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2. The cells were plated onto sterilized glass coverslips in 35-mm dishes prior to transfection. Transient transfection of 1 μg of DNA was carried out using Effectene (Qiagen) for MDCK cells and FuGENE 6 (Roche Applied Science) for HeLa cells. For translocation experiments, 0.1 μg of MyrPalmCFP or CFP-NHERF-1 DNA was used in transient transfections. The cells were imaged within 24 h following transfection. The cells were washed once in Hanks' balanced salt solution (Cellgro) and imaged in Hanks' balanced salt solution in the dark at room temperature. The cells were stimulated with 200 nm PDBu, 10 μm histamine, 250 nm Gö 6976, or 500 nm Gö 6983 where indicated. CFP, YFP, and FRET images were acquired and analyzed as described (21.Kunkel M.T. Ni Q. Tsien R.Y. Zhang J. Newton A.C. J. Biol. Chem. 2005; 280: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). mCherry was visualized through a 560/25-nm excitation filter, a 593-nm dichroic mirror, and a 629/53-nm emission filter. HeLa cells were grown to 80% confluency in 60-mm dishes and then transfected using Lipofectamine 2000 (Invitrogen) with 20 nm control or NHERF-1 SMARTpool siRNA (Dharmacon). 48 h post-transfection the cells were rinsed in phosphate-buffered saline and then treated for the indicated times with 10 μm histamine at room temperature. The cells were lysed in 50 mm Na2HPO4, 1 mm Na4P2O7, 20 mm NaF, 2 mm EDTA, 2 mm EGTA, 1% Triton X-100 (supplemented with 1 mm dithiothreitol, 200 μm benzamidine, 40 μg ml−1 leupeptin, 300 μm phenylmethylsulfonyl fluoride, and 1 μm microcystin) and cleared by a high speed spin. The cleared lysates were analyzed by Western blotting to determine the relative amount of NHERF-1, phosphorylated PKD substrates, and actin. The Western blots were developed using chemiluminescence and quantified using Image J (National Institutes of Health). The last four amino acids of both PKD1 and PKD2 (VSIL and ISVL, respectively) comprise a PDZ-binding motif. To identify PDZ domains with which these kinases could interact, a GST fusion protein of the last 25 residues of either PKD1 or PKD2 was overlaid onto a membrane array spotted with 96 unique PDZ domains (20.Fam S.R. Paquet M. Castleberry A.M. Oller H. Lee C.J. Traynelis S.F. Smith Y. Yun C.C. Hall R.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8042-8047Crossref PubMed Scopus (82) Google Scholar). Binding was detected with GST antibodies. Fig. 1 shows that the PDZ-binding motifs of both PKD1 and PKD2 interact with the first PDZ domain of NHERF-1 (spot B3, NHERF-1 PDZ1). In addition, the PDZ-binding motif of PKD2 bound the fifth PDZ domain of MAGI-3 (spot B2, MAGI-3 PDZ5). No detectable binding was observed when GST alone was overlaid on the array (data not shown). Having identified interactions of the isolated PDZ-binding motifs of PKD1 and PKD2 with NHERF-1, we next addressed whether the full-length proteins could interact in cells. We focused on examining potential cellular associations between the PKD isoforms and NHERF-1 using FRET (Fig. 2A) rather than traditional biochemical approaches, for the following reasons: 1) PDZ domain interactions with PDZ-binding motifs are often weak in nature (22.Chen J.R. Chang B.H. Allen J.E. Stiffler M.A. MacBeath G. Nat. Biotechnol. 2008; 26: 1041-1045Crossref PubMed Scopus (121) Google Scholar), and 2) the serines within the PDZ-binding motifs of both PKD1 and PKD2 (VSIL and ISVL of PKD1 and PKD2, respectively) are known to become phosphorylated following PKD activation, which would likely destroy the PDZ-binding motifs and further contribute to the transient nature of PKD/NHERF-1 interactions. Because FRET is measured in real time and only occurs when the fluorophores (CFP and YFP in this instance) are within 10 nm of each other, the use of FRET is ideal for allowing the visualization of transient cellular interactions between proteins (23.Sekar R.B. Periasamy A. J. Cell Biol. 2003; 160: 629-633Crossref PubMed Scopus (655) Google Scholar). CFP-NHERF-1 was co-expressed with either YFP-PKD1 or YFP-PKD2 in MDCK cells, and the change in FRET (plotted as FRET/CFP) was monitored over time following stimulation of the PKD pathway. The data in Fig. 2 (B and C) show that PDBu caused a robust increase in the FRET/CFP ratio for PKD1 and PKD2, revealing translocation of both isoforms to NHERF-1. Phorbol ester-triggered translocation was rapid and transient, peaking within 1 min and then decaying with a half-life of approximately 2 min. This decay of the signal may reflect autophosphorylation at the serine within the PDZ-binding motif of each kinase, because this autophosphorylation event is known to occur with similar kinetics (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Interestingly, FRET between the PKDs and NHERF-1 decreased to levels below the original base line, suggesting that there was some PKD pre-associated with NHERF-1 prior to PDBu treatment. To verify that this association was dependent on the PDZ-binding motif of the kinase, we generated YFP-PKD2 with its PDZ-binding motif deleted (YFP-PKD2ΔPDZ). No significant changes in FRET were observed following PDBu treatment in cells co-expressing YFP-PKD2ΔPDZ and CFP-NHERF-1 (Fig. 2C, open diamonds). Importantly, all YFP kinase proteins translocated to and remained at the plasma membrane over the same time frame in FRET experiments using MyrPalmCFP as the FRET donor (Fig. 2, D and E). We next visualized the co-localization of PKD2 and NHERF-1 in cells by fluorescence microscopy. Fig. 2F shows that the CFP-NHERF-1 signal is localized to apical membranes in MDCK cells, as described previously (24.Georgescu M.M. Morales F.C. Molina J.R. Hayashi Y. Curr. Mol. Med. 2008; 8: 459-468Crossref PubMed Scopus (82) Google Scholar), and remains relatively stable over the course of the experiment (left column). In contrast, YFP-PKD2 is present in the cytosol prior to PDBu addition (Fig. 2F, middle column, top panel) and is predominantly at membranes bound to PDBu by the end of the experiment (Fig. 2F, middle column, bottom panel). In support of the FRET data, a high degree of co-localization is apparent after 1 min of PDBu treatment, i.e. when maximal FRET is observed in Fig. 2C (Fig. 2F, middle column, middle panel). This co-localization is further revealed by the yellow signal in the merged images on the right column of Fig. 2F. To explore whether the interaction of the PDZ-binding motifs of PKD1 and PKD2 with NHERF coordinates PKD signaling at NHERF scaffolds in cells, we took advantage of a genetically encoded PKD activity reporter, D kinase activity reporter (DKAR) (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), to examine PKD activity at the scaffold. DKAR consists of the FRET pair mCFP (monomeric cyan fluorescent protein) and mYFP (the monomeric version of the yellow fluorescent protein citrine (25.Griesbeck O. Baird G.S. Campbell R.E. Zacharias D.A. Tsien R.Y. J. Biol. Chem. 2001; 276: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar)) flanking an FHA2 phosphothreonine-binding domain and a consensus PKD phosphorylation sequence. Phosphorylation of the substrate sequence triggers an intramolecular clamp with the phosphopeptide-binding domain, leading to a change in FRET (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Both PKD1 and PKD2 efficiently phosphorylate DKAR and induce a change in FRET (data not shown). Because it is a genetically encoded reporter, DKAR can be effectively targeted to distinct subcellular compartments by the addition of short targeting sequences (26.Gallegos L.L. Kunkel M.T. Newton A.C. J. Biol. Chem. 2006; 281: 30947-30956Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Thus, to poise DKAR at the NHERF scaffold to assess localized PKD activity, we designed a reporter with a PDZ-binding motif that would target to NHERF but not necessarily compete with, and potentially disrupt, the interaction of endogenous PKD1 or PKD2 with NHERF-1. To this end, we chose a distinct PDZ-binding motif from the phosphatase PHLPP2 (17.Brognard J. Sierecki E. Gao T. Newton A.C. Mol. Cell. 2007; 25: 917-931Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar) that interacts with both the first and second PDZ domain of NHERF-1 and both PDZ domains of the interacting NHERF-2 (data not shown). To test whether this PDZ-binding motif (which comprises the sequence DTAL) targets to NHERF-1, we expressed a construct of CFP fused to the carboxyl-terminal 10 residues of PHLPP2 in MDCK cells and imaged its localization. Expression of this CFP-PDZ fusion protein alone revealed diffuse cellular staining (Fig. 3A), consistent with no specific targeting. In contrast, co-expression with mCherry-NHERF-1 (Fig. 3B) revealed significant localization of CFP-PDZ to mCherry-NHERF-1 at apical membranes where the NHERF scaffold localizes (24.Georgescu M.M. Morales F.C. Molina J.R. Hayashi Y. Curr. Mol. Med. 2008; 8: 459-468Crossref PubMed Scopus (82) Google Scholar). Having identified a PDZ-binding motif distinct from that of PKD1 or PKD2 that targets to NHERF-1, we fused this motif onto DKAR to generate DKAR-PDZ. MDCK cells overexpressing DKAR or DKAR-PDZ with or without overexpression of NHERF-1 were analyzed following stimulation with the phorbol ester PDBu, and the ratio of cyan to yellow emission (FRET ratio) was monitored. The FRET ratio from DKAR (Fig. 4, open circles) and DKAR-PDZ (Fig. 4, open diamonds) increased to similar levels following PDBu treatment. Overexpression of the untargeted DKAR and NHERF-1 gave a similar response (Fig. 4, filled circles). However, co-expression of DKAR-PDZ and NHERF-1 resulted in a significantly enhanced FRET ratio change following PKD activation with PDBu (Fig. 4, filled diamonds); the amplitude of the FRET change increased 2.5-fold. Interestingly, the fluorescent signal from DKAR-PDZ with NHERF-1 overexpressed showed modest, if any, membrane localization (data not shown); however, the increased PKD activity reported by DKAR-PDZ in cells overexpressing NHERF-1 reveals relocalization of the reporter to the scaffold. Note that it is possible that the PDZ ligand on DKAR-PDZ competes some of the endogenous PKD from NHERF-1; however, given the robust and unique DKAR signature at the NHERF scaffold, it is clear that there is sufficient localized PKD to catalyze readily detectable phosphorylation of the reporter poised at NHERF. If anything, the reporter might under-report the true magnitude of PKD activity at the scaffold. Although DKAR-PDZ is not dramatically relocalized when NHERF-1 is co-expressed, the increased PKD activity observed from the reporter in this context could reflect membrane association, because NHERF-1 is predominantly membrane-associated. To verify that the change in the FRET ratio reflected PKD activity at NHERF-1 as opposed to PKD activity at the plasma membrane, we compared the DKAR signal for reporter scaffolded to NHERF-1 compared with reporter scaffolded to general plasma membrane. Plasma membrane targeting was achieved by the addition of the seven amino-terminal residues derived from Lyn kinase that encode for the myristoylation and palmitoylation of DKAR (MyrPalmDKAR) (16.Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1790) Google Scholar). Treatment of MDCK cells co-expressing MyrPalmDKAR and NHERF-1 (Fig. 5A, filled squares) with PDBu resulted in an almost 2-fold reduction in the amplitude of the FRET ratio change compared with MDCK cells co-expressing DKAR-PDZ with NHERF-1 (Fig. 5A, filled diamonds). Interestingly, expression of MyrPalmDKAR alone, in the absence of NHERF-1 overexpression, resulted in a significant increase in DKAR phosphorylation, suggesting that the scaffold sequestered PKD to NHERF-1 and away from MyrPalmDKAR. These data reveal increased PKD activity at NHERF scaffolds relative to activity at the plasma membrane under conditions where the NHERF-1 protein is overexpressed. DKAR phosphorylation is reversible, allowing the reporter to read both PKD activation and deactivation (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To further explore differences in the signature of PKD activity at the NHERF scaffold compared with the plasma membrane, we treated actively signaling cells with the PKD inhibitor Gö 6976 to monitor the rate of DKAR dephosphorylation, in the context of PKD inactivation. Cells expressing DKAR-PDZ with NHERF-1, MyrPalmDKAR with NHERF-1, or MyrPalmDKAR alone were treated with PDBu for 15 min to allow DKAR phosphorylation to plateau, and then cells were treated with the PKD inhibitor. The graph in Fig. 5B shows that inhibition of PKD resulted in rapid dephosphorylation of DKAR-PDZ (filled diamonds) compared with the slow dephosphorylation of MyrPalmDKAR (squares). Thus, the local phosphatase environment around DKAR-PDZ poised at NHERF-1 is relatively high and distinct from the low level of phosphatase activity at the plasma membrane. This distinct response from MyrPalmDKAR indicates that DKAR-PDZ is in fact poised at the NHERF-1 scaffold, and its activity is not simply reflecting PKD signaling at the general plasma membrane. Having established that PKD activity is enriched at NHERF-1 using the potent PKD activator PDBu, we next explored PKD signaling at the scaffold following stimulation of endogenous signaling pathways. Histamine has been shown to activate G protein-coupled receptors in HeLa cells, resulting in the activation of numerous signaling molecules including PKD (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). As described previously, the DKAR response to histamine in HeLa cells is rapid and begins to decay within 2 min (14.Kunkel M.T. Toker A. Tsien R.Y. Newton A.C. J. Biol. Chem. 2007; 282: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Here we expressed DKAR or DKAR-PDZ in HeLa cells with or without NHERF-1 and activated PKD signaling via stimulation of endogenous histamine receptors. The response from DKAR with NHERF-1 or from DKAR-PDZ alone showed a similar transient profile as from DKAR alone (Fig. 6A). However, the FRET response observed from DKAR-PDZ following histamine treatment was significantly different when NHERF-1 was co-expressed; the phosphorylation of DKAR-PDZ was sustained and, additionally, reached a lower initial amplitude than the untargeted reporter (Fig. 6A, filled diamonds). This smaller but prolonged response reflects tight regulation of PKD phosphorylation at NHERF-1 because further PKD stimulation by PDBu did not significantly increase the FRET ratio change, and PKD inhibition by Gö 6976 did not reverse the FRET ratio beyond its base-line value (data not shown). To verify that the response from DKAR-PDZ with NHERF-1 to histamine was a readout of PKD activity at NHERF-1 as opposed to PKD activity at the plasma membrane, we again utilized MyrPalmDKAR and assessed signaling by PKD at plasma membranes. The FRET response from MyrPalmDKAR in HeLa cells treated with histamine displayed strikingly distinct kinetics from DKAR-PDZ at NHERF-1; the rate of phosphorylation was reduced 8-fold from a half-time of approximately 1 min at the scaffold (Fig. 6A, filled diamonds) to a half-time of ∼8 min at the plasma membrane (Fig. 6B, filled squares). In addition, the amplitude of the signal was 3-fold higher for MyrPalmDKAR compared with DKA" @default.
• W2083021738 created "2016-06-24" @default.
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• W2083021738 creator A5067712501 @default.
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• W2083021738 creator A5083622095 @default.
• W2083021738 date "2009-09-01" @default.
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• W2083021738 title "The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D Activity" @default.
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