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J. Biol. Chem., Vol. 281, Issue 26, 17801-17814, June 30, 2006
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11
2
From the
Department of Molecular Pharmacology, Atran Laboratories and the Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, November 4, 2005 , and in revised form, April 5, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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60% of normal length. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
, and 
(1–5). PKCs phosphorylate specific Ser/Thr hydroxyl groups in substrate/effector proteins, thereby modulating ion transport, secretion, cell growth, differentiation, gene transcription, and other physiological processes. Aberrant activation of PKCs is linked to tumor promotion, myocardial hypertrophy and infarction, and pathophysiological complications of type II diabetes (6–11). Upon PLC activation, diacylglycerol (DAG) binding modules (C1 domains) ligate membrane-intercalated DAG. Consequently, PKCs translocate from cytoplasm to a membrane surface enriched in phosphatidylserine (PS), a PKC activator. Association of PKCs with DAG and PS elicits expulsion of the pseudosubstrate domain (12) from the catalytic cleft and enables expression of intrinsic kinase activity (3, 5, 13, 14). Ligand binding with >100 types of cell surface receptors triggers production of DAG in numerous mammalian cells. Knowledge of structures, activation mechanisms, and intracellular trafficking of PKCs is extensive. In contrast, information about in situ regulation, downstream effectors, interacting modulatory proteins, and the precise physiological roles of individual PKC isoforms is more limited.
The discovery of protein kinase D (PKD) isoforms (PKD, PKD2, and PKD3) revealed a new dimension in DAG-mediated signal transduction (15–18). PKDs are composed of 900 amino acids and contain tandem C1 domains and a Ser/Thr protein kinase module near their N- and C termini, respectively. PMA or DAG promotes redistribution of PKDs from cytoplasm to membranes and concomitant activation of PKD catalytic activity (19–21). PKDs are distinguished from PKCs by (a) the presence of a pleckstrin homology (PH) domain, (b) the absence of a pseudosubstrate site, and (c) divergent substrate specificity (18–23). Application of PKC-selective inhibitors and expression of constitutively active PKCs in transfected cells has disclosed that PKCs govern PKD activation (24–26). Thus, PKDs are candidate physiological effectors for DAG-stimulated PKCs.
In mammals, PKD activation is associated with cell replication, tumorigenesis, Golgi vesicle fission and trafficking, adhesion, responses to stress, NF
B activation, modulation of JNK activity, and other processes (5, 19–21). Current concepts of regulation and physiological consequences of PKD activation are based predominantly on expression of wild type (WT) and mutant PKDs expressed in transiently transfected cells. Important insights into the properties of PKDs were acquired via transient transfection analysis, but inherent limitations of the methodology merit consideration. Typically, supraphysiological levels of WT/mutant PKDs accumulate in transfected cells. Thus, the potential for (a) mislocalization of activated or inhibitory ("dominant negative," "kinase dead") PKDs, (b) promiscuous phosphorylation of minor or nonrelevant substrates, and/or (c) association of PKD with atypical binding partners is increased. High level PKD expression may ablate counter-regulatory processes such as effector dephosphorylation or hinder operation of negative feedback loops. If these factors apply, data interpretation could be compromised and predictions of PKD physiological roles might be qualitatively or quantitatively imprecise.
Individual domains may specify subcellular location, level of catalytic activity, nuclear entry and exit, membrane association, or other attributes of PKDs (27–33). However, reports of complex or contradictory structure/function relationships have also confounded straightforward assignment of particular roles to specific domains in several instances. For example, both PKD that is diphosphorylated at Ser residues in the activation loop and PKD lacking phospho-Ser in the same loop are proposed as candidate mediators of NFB activation (25, 34–36). Thus, a lack of information derived from (a) direct assessment or depletion of endogenous PKDs or (b) expression of near physiological levels of WT and mutant D kinases precludes definitive answers to the question of whether observed DAG/PKC/PKD signaling pathways are major or auxiliary (cell-specific or ubiquitous, etc.) contributors to overall cell/organism physiology. Full comprehension of effects of PKDs in homeostasis and hormone/GF actions will require more advanced knowledge of the complex regulatory interplay among PKD and PKC isoforms, rigorous analysis of modes of PKC-independent PKD regulation/activation, and discovery of critical physiological changes in vivo that are specifically linked to alterations in PKD concentration and/or activity. At present, many central questions about PKDs remain unresolved including the following. 1) Are all PKD isoforms PKC effectors? 2) Can PKDs independently receive, amplify, and disseminate signals carried by DAG? 3) Are individual PKDs indispensable for specific physiological processes in intact animals? If so, which functions? 4) Will targeted overexpression in vivo reveal novel physiological roles for PKDs?
Answers to the posed questions may be acquired by coupling investigations on mammalian systems with complementary studies on PKDs in a model organism. Caenorhabditis elegans is an attractive choice because its cell biology, development, and physiology are characterized in exceptional detail (37–43). Moreover, C. elegans employs signaling molecules, mechanisms, and pathways that are conserved among eukaryotes (44–49). Techniques for gene disruption, RNA interference (RNAi), and targeted mRNA/protein expression in specific cells are optimized for in vivo analysis in C. elegans (50–54). Typically, information and insights derived from experimentation on C. elegans signal transduction networks complement and synergize with data and concepts obtained from investigations on parallel signaling pathways in mammals (39, 41, 44–49). We now report the discovery, detailed characterization, and consequences of in vivo depletion of a novel D kinase family isoform (DKF-1). DKF-14 is a new prototype within the PKD family. In an accompanying article (89), we report on a series of unique regulatory properties in the C1a, C1b, PH, and kinase domains of DKF-1.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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gt10 bacteriophage as described by Hu and Rubin (55). Positive cDNA inserts were excised from recombinant phages and cloned in the plasmid pGEM7Z (+) (Promega). One partial cDNA insert was repeatedly retrieved. Consequently, this (1.6 kbp) cDNA was excised by digestion with EagI and EcoRV, purified, and radiolabeled by random priming. A C. elegans cDNA library in bacteriophage ZAPII (Stratagene) was hybridized with the 32P-labeled cDNA fragment. pBluescript SK phagemids that contained hybridizing cDNA inserts were excised from phage in Escherichia coli (in situ), amplified, and purified. Cloned cDNAs (2.3 kbp), which contain the complete coding sequence and 3'-untranslated region of DKF-1 mRNA, were isolated. DNA Sequencing and Analysis—Genomic and PCR-amplified DNA and DKF-1 cDNAs were sequenced as described previously (56). Sequence comparisons and data base searches were performed with programs provided by NCBI servers at National Institutes of Health, Bethesda, MD. Protein domains were identified and characterized by using the SMART Web site (European Molecular Biology Laboratories, Heidelberg, Germany) (57). ClustalW programs were accessed at European Bioinformatics Institute, Hinxton, UK.
Purification of DKF-1 Fusion Protein Expressed in Escherichia coli—A DKF-1 cDNA fragment encoding amino acids 2–154 (Fig. 1A) was synthesized via PCR as reported previously (58, 59) using the high fidelity DNA polymerase PfuTurbo (Stratagene). Unique EcoRI (5') and XhoI (3') restriction enzyme sites were incorporated into the PCR primers. Product DNA was digested with EcoRI and XhoI and cloned into the bacterial expression plasmid pGEX-3X (GE Healthcare). Vector DNA encoding Schistosoma japonicum glutathione S-transferase (GST) precedes the cDNA insert. Fusion gene transcription/translation is controlled by an isopropyl-1-thio--D-galactopyranoside (IPTG)-inducible tac promoter. Subsequent steps in fusion protein production are described in detail by Land et al. (60). Approximately 1.5 mg of highly purified DKF-1 fusion protein was isolated from a 1-liter culture of E. coli.
Production of Antiserum Directed against DKF-1—DKF-1 fusion protein was injected into rabbits (an 0.4-mg initial injection; 0.2 mg for each of four booster injections) at Covance Laboratories (Vienna, VA) at 3-week intervals. Antiserum was collected at 3-week intervals.
Affinity Purification of Anti-DKF-Immunoglobulins (IgGs)—Three milligrams of GST-DKF-1 (2–154) was coupled to 2 ml of Sepharose 4B (GE Healthcare) as described previously (61). Anti-DKF-1 IgGs were then purified from 2 ml of immune serum by affinity chromatography as published previously (60, 62). Purified IgGs were dialyzed against phosphate-buffered saline containing 50% glycerol and stored at –20 °C.
Growth and Synchronization of C. elegans—Bristol N2 WT C. elegans was grown, synchronized, harvested, and pulverized into powder in a mortar cooled with liquid N2. L1 larvae were harvested 6 h after hatching, L2 larvae at 20 h, L3 larvae at 29 h, L4 larvae at 40 h, and young adult C. elegans at 52 h; egg-laying adult animals were collected at 78 h. A complete description of the procedures is given in Hu and Rubin (55).
Preparation of Particulate and Cytosolic Fractions from C. elegans—Frozen, powdered nematodes were suspended in buffer and disrupted as reported previously (60). Homogenates were centrifuged at 100,000 x g to separate and isolate supernatant solution (cytosol) and an insoluble pellet fraction. Cytosol and resuspended pellet were stored in liquid nitrogen. Details are provided in Ref. 60.
In Vitro Synthesis of DKF-1—A coupled transcription-reticulocyte lysate translation system (TNTTM, Promega Corp.) was programmed with 1 µg of recombinant pBluescript that contains full-length DKF-1 cDNA. Incubation, denaturing electrophoresis, and autoradiography were performed as published previously (55, 62).
Cell Culture—A cell line derived from a hamster subcutaneous tumor (AV-12) and human HEK293 cells were obtained from the American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in an atmosphere of 7% CO2 and 93% air.
Electrophoresis of Proteins and Western Immunoblot Assays—Cytosol and particulate proteins were isolated from mammalian cells as stated previously (63). Triton X-100 was added or omitted from cell lysis buffer as indicated in the legend for Fig. 4 and under "Results." Proteins were size-fractionated by electrophoresis in a denaturing polyacrylamide (8% or otherwise indicated) gel as reported previously (64). BenchMarkerTM prestained proteins (9–182 kDa, Invitrogen) or Precision Plus ProteinTM (10–250 kDa, Bio-Rad) polypeptides were used as molecular size standards. Western blots of size-fractionated proteins were prepared, blocked, incubated with anti-DKF-1 IgGs (1:1000), and washed as indicated in previous studies (63, 64). Unless noted otherwise, each lane in Western blots received 30 µg of protein. Antigen·antibody complexes were visualized and quantified by using an enhanced chemiluminescence procedure and image analysis software (ImageQuant, GE Healthcare).
Purification of DKF-1 Fusion Protein Expressed in Sf9 Cells—Full-length DKF-1 cDNA was cloned downstream from a GST gene in a baculovirus transfer vector (pAcGHLT, Pharmingen). Recombinant baculovirus encoding GST·DKF-1 was generated and amplified as described in Islas-Trejo et al. (65). Procedures for infection, growth, and lysis of Sf9 cells are elaborated in detail elsewhere (60, 65). Fusion protein was purified by affinity chromatography on GSH-Sepharose 4B (60). Fractions containing high levels of DKF-1 were pooled and stored at –80 °C.
Expression of DKF-1 in Mammalian Cells—Full-length DKF-1 cDNA, excised from recombinant pBluescript plasmids (see above) by digestion with XbaI and Asp718, was cloned in the mammalian expression vector pCDNA3.1(+) (Invitrogen, Carlsbad CA). Hamster AV-12 or human HEK293 cells were transfected with the recombinant DKF-1 transgene using LipofectamineTM (Invitrogen). Stable cell lines that express modest amounts of DKF-1 protein were obtained by selection with 1 mg/ml G418 for 14 days. Individual drug-resistant clones were expanded and verified by independently determining DKF-1 protein levels and measuring PMA-stimulated kinase activity in cell extracts (see below).
Protein Determination—Protein concentrations were determined with the Bio-Rad DC protein assay reagents. Bovine albumin was employed as a standard.
Immunoprecipitation and in Vitro DKF-1 Kinase Assays—AV-12 cells were transfected by calcium phosphate precipitation (58, 63) or uptake of LipofectamineTM-recombinant DNA complexes. After 24 h, cells were harvested and homogenized in lysis buffer as reported previously (63). All operations (except kinase assays) were performed at 4 °C. Cell lysates were centrifuged at 40,000 x g for 30 min, and samples of supernatant solution (0.1–0.3 mg of protein) received 2 µl(1 µg) of affinity-purified anti-DKF-1 IgG. Incubation for 3 h yielded an optimal level of antigen·IgG complex formation. Subsequently, 25 µl of protein A (or G)-Sepharose 4B beads (Zymed Laboratories Inc.) was added, and the incubation was continued for 60 min. Next, bead-bound immune complexes were washed three times with lysis buffer and then twice with kinase buffer containing 25 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and 1 mM dithiothreitol.
Catalytic activity of DKF-1 was quantified by measuring incorporation of 32P radioactivity from [-32P]ATP into Syntide-2 peptide substrate (Calbiochem). Reaction buffer (30 µl, containing 25 mM Tris-HCl, pH 7.4, 30 µM peptide substrate, 100 µM [-32P]ATP (150 cpm/pmol), 5 mM MgCl2, 0.5 mM EGTA, and 1 mM dithiothreitol) was added to immune complexes, and samples were incubated at 30 °C for 10 min. Assays were terminated by the addition of 5 µl of 0.2 M EDTA, pH 8.0, containing 60 mM NaF. Reaction mixtures were applied to P81 filter papers (Whatman) and subjected to extensive washing in 75 mM H3PO4 (32P-Syntide-2 binds P81 filters under acidic conditions, whereas 32Pi and [-32P]ATP are washed away). After filters were washed and air-dried, 32P radioactivity incorporated into Syntide-2 was measured in a scintillation counter. Phosphotransferase activity of purified GST·DKF-1 fusion protein from Sf9 cells was assayed as described above, except that 3 µl of enzyme (50 ng protein) was used instead of an immunoprecipitate. Various combinations of PS, DAG, and Ca2+ were added to kinase buffer to test their effects on catalytic activity.
PKC peptide RFARKGSLRQKNV, PKC
peptide YRRGSRRWKKIY, Syntide-2 peptide PLARTLSVAGLPGKK, an optimum PKD peptide designed by Cantley et al. (23), AALVRQMSVAFFF, and myelin basic protein were tested as substrates for DKF-1. Potential phosphorylation sites are shown (above) in bold underlined italics.
To test the effects of PKC inhibitors on DKF-1 catalytic activity in situ, AV-12 cells were incubated with various concentrations of bisindolylmaleimide I (GF109203X) or bisindolylmaleimide IX (RO31-8220) (Axxora, LLC) for 1 h. Cells were then treated with specified concentrations of PMA for 10 min. In vitro kinase assays were performed on immunoprecipitated DKF-1 as described above. PKC inhibitors were tested in vitro by adding various amounts of GF109203X or RO31-8220 directly to the kinase reaction mixture.
Single Worm PCR Amplification of a Disrupted dkf-1 Gene—Deletions of 1–3 kbp in target genes were generated by mutagenesis with Psoralen and ultraviolet light by LiaoTeng Wang in the laboratory of Dr. Judith Kimble (Department of Genetics, University of Wisconsin). Individual adult worms from potential dkf-1 knock-out lines were extracted by incubation with 5 µl of lysis buffer (50 mM KCl, 10 mM Tris-HCl, PH 8.2, 2.5 mM MgCl2, 0.45% Nonidet P-40, 0.45% Tween 20, 0.01% gelatin, and 5% proteinase K) at 65 °C for 1 h. Large fragments of the dkf-1 gene were amplified by nested PCR using genomic DNA in diluted C. elegans extracts as template. PCR was first executed with an "external left" primer (5'-TCCGAAGAAGATGATCCAGG-3') and an "external right" primer (5'-CAATTGTGCAATCACGCTCC-3') pair. A second round of PCR used internal left (5'-AAACACAGTTCGAAGCGAGC-3') and internal right (5'-AACTGCCACCCAATTTCAGG-3') primers. Sizes of amplified DNA segments were estimated by electrophoresis in a 1% agarose gel. Exact deletion boundaries were determined by sequencing the amplified genomic DNA.
Preparation of Transgenic C. elegans—A DNA fragment (2.5 kbp) that precedes the 5' end of the dkf-1 structural gene was ligated to cDNA encoding the complete DKF-1 open reading frame in pBluescript SK by standard procedures. Subsequently, the fusion DNA (promoter + open reading frame) was cloned into pPD 95.77, a C. elegans expression vector (52). A DKF-1 cDNA translation stop codon was eliminated by mutagenesis to permit in-frame ligation with DNA encoding green fluorescent protein (GFP). The GFP coding region is followed by translation termination and poly(A) addition signals. DKF-1 promoter/enhancer DNA ensures normal temporal developmental and cell-specific regulation and expression of DKF-1·GFP protein. Fluorescence signals emanating from individual cells of living or fixed worms are recorded via microscopy systems equipped for epifluorescence.
C. elegans was transformed by microinjecting the DKF-1·GFP kinase-reporter plasmid (dkf-1P::DKF-1·GFP) as described previously (66). Transgenic C. elegans were identified by fluorescence microscopy and transferred to new plates to establish cloned populations. The dkf-1P::DKF-1·GFP chimeric gene was chromosomally integrated as described in a protocol provided by Dr. Michael Koelle (Department of Molecular Biophysics and Biochemistry, Yale University). Expression of DKF-1·GFP in C. elegans allows visualization of authentic promoter activation in individual cells in vivo. Fluorescence signals were captured as reported previously (60, 65).
Ablation of DKF-1 Protein and Function by RNAi—Full-length DKF-1 cDNA was cloned into PPD129.36 (generously provided by Dr. A. Fire, Departments of Pathology and Genetics, Stanford University School of Medicine), a vector that has two opposing promoters for T7 RNA polymerase on sense and antisense DNA strands (67–69). E. coli HT115 (DE3) was transformed with recombinant plasmid, grown to A595 = 0.4 and seeded on plates containing 0.8 mM IPTG. L4-stage worms were transferred to plates containing transformed bacteria. Double-stranded RNAi ingested by C. elegans is disseminated to cells throughout the nematode via double-stranded RNA transporters (67–69). After 30–36 h at 20 °C, individual adult worms were transferred to fresh plates, and phenotypes of adults and offspring were observed by light microscopy.
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RESULTS |
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81,000. In vitro transcription/translation programmed by the 2.3-kbp cDNA yielded an 81-kDa polypeptide (Fig. 2D). This polypeptide has the same apparent Mr and shares epitopes with DKF-1 protein that (a) accumulates in cultured cells expressing a DKF-1 transgene (Fig. 2C) and (b) is abundant (endogenously) in vivo in a subset of cells in C. elegans (Figs. 2, B and E, 7, and 8, A and D).
Structure/Function Relationships in DKF-1—A C-terminal region (amino acids 426–685, Fig. 1A) of DKF-1 contains sequence motifs that are conserved in catalytic domains of Ser/Thr protein kinases. Functions for these conserved amino acids were established by biochemical analysis, mutagenesis, comparisons with sequences of several hundred phosphotransferases, and insights derived from crystal structures of prototypic Ser/Thr protein kinases (71–74). Sequences in DKF-1 are tentatively linked to specific functions by analogy. A GXGXXGX16K motif (residues 433–455, Fig. 1A) provides sites that bind with phosphates of the substrate MgATP. Lys455 is essential for catalysis and binding of ATP. A DFG tripeptide (residues 573–575, Fig. 1A) contributes a carboxyl side chain (Asp573) that can mediate binding of divalent metal and phosphate of MgATP and stabilize pentavalent phosphorous in the transition state for the kinase reaction. Glu599, which is part of a conserved PPE motif (APE in other protein kinases), as well as Asp611 and Arg673, stabilizes the catalytic core region. An XXDLKXX(N/D) motif is a Ser/Thr protein kinase "signature" sequence that guides protein substrate into an orientation favorable for catalysis (71–74). The DKF-1 catalytic loop sequence, HCDLKPEN (residues 549–556), diverges from the corresponding loop (YRDLKLDN) present in all members of the related PKC family. However, the HCDLKPEN motif is conserved among PKD isoforms (15–18).
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In PKC isoforms, a pseudosubstrate motif (Ala flanked by basic amino acids) is located 15 amino acids upstream from the first C1 domain (C1a). DKF-1 lacks a classical pseudosubstrate sequence. Unlike PKCs, DKF-1 contains a PH domain (residues 279–407, Fig. 1A), which is 33% identical (50% similarity) with an analogous domain in mammalian PKD. DKF-1 also lacks a calcium-binding domain (C2) that is conserved in cPKC isoforms (Fig. 1B).
Sequence Conservation and Divergence between DKF-1 and Human PKD Isoforms—The amino acid sequences of DKF-1 and protein kinases in standard data bases were aligned. DKF-1 is most closely related to mammalian PKD, PKD2, and PKD3 isoforms (50% similarity; 
38% identical amino acids with each isoform) (Table 1). No other protein kinases share >26% overall identity with DKF-1. Sequence homology varies markedly among domains (Table 1). The catalytic domains of human PKD isoforms and nematode DKF-1 are maximally conserved (60% identity). DKF-1 and PKDs also share high levels of homology in the C1a and C1b regulatory regions (44–58% identity, Table 1). Four His and 12 Cys residues, which are crucial for organizing/stabilizing zinc finger structures and binding physiological activators (DAG, PS) to C1 domains of PKCs and PKDs, are retained in perfect register in DKF-1. The PH domain and extreme C-terminal region of DKF-1 have lower but significant levels of sequence identity with corresponding segments in human PKDs (30–33% identity plus 20% conserved amino acid substitutions, Table 1). Conservation of amino acid sequences and the relative positions of domains along the polypeptide chain (Fig. 1B, Table 1) indicate that DKF-1 is a member of the PKD family. However, major differences in structure/function relationships and mode of activation (elaborated in detail below and in an accompanying article (89)) indicate that DKF-1 is a novel, previously uncharacterized PKD isotype.
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Organization of the C. elegans dkf-1 Gene—During the course of our investigations on DKF-1, the C. elegans Genome Project deposited genomic DNA sequence data for cosmid WO9C5 in GenBankTM and Wormbase. We established the organization of exons and introns for the dkf-1 structural gene by identifying genomic DNA sequences in recombinant cosmids that overlap with segments of DKF-1 cDNA. Our results and subsequent analysis by the C. elegans Genome Consortium are in agreement (Table 2). The dkf-1 structural gene contains 11 exons and spans 8 kbp of DNA (Table 2).
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Regulation of DKF-1 Expression during Development—Cytosolic and total particulate proteins were isolated from C. elegans at each stage of development. Western immunoblot analysis revealed that DKF-1 is expressed robustly in embryos. Lower but substantial amounts of the kinase are present as animals progress through four larval stages and adulthood (Fig. 2E). DKF-1 is isolated in the cytosolic fraction of C. elegans homogenates (Fig. 2E). DKF-1 protein accumulates principally in the cytoplasm of (a) AV-12 cells carrying a constitutively expressed dkf-1 transgene and (b) Sf9 cells infected with recombinant baculovirus that contains a dkf-1 cDNA insert (data not shown).
Purification and Properties of Recombinant DKF-1—A preparation of DKF-1 that is free of other PKDs, PKC isoforms, and unrelated but potentially interfering protein kinases was required for unequivocal characterization of the novel C. elegans kinase. Consequently a chimeric gene, encoding full-length DKF-1 polypeptide fused (in-frame) to the C terminus of GST, was created by inserting dkf-1 cDNA into a baculovirus transfer/fusion vector, pAcGHLT. Recombinant virus was generated and used to infect Sf9 cells. Cytosol was isolated 4 days after infection, and DKF-1 was purified by affinity chromatography. Purified DKF-1 fusion protein exhibited modest basal activity in the absence of co-factors. Phosphorylation of Syntide-2 (PLARTLSVAGLPGKK), a preferred substrate for mammalian PKDs, was only minimally increased by DAG or PS alone. However, a combination of PS and DAG synergistically increased DKF-1 phosphotransferase activity 15–20-fold (Table 3). Evidently, simultaneous binding of both co-factors is crucial for establishing/maintaining maximal kinase activity. DKF-1 phosphotransferase activity was not influenced by calcium which stimulates conventional PKCs (Table 3). Native nonfused DKF-1 was isolated and purified from extracts of transiently or stably transfected cells by immunoprecipitation. In vitro kinase assays performed with native or recombinant DKF-1 yielded essentially the same results under all conditions.
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and PKC
pseudosubstrate peptides, in which Ser substitutes for Ala, are poor phospho-acceptors for DKF-1. A model small protein substrate, myelin basic protein, which is phosphorylated by many PKC isoforms and other regulatory kinases, is not a good target for DKF-1 (Fig. 3A). Using combinatorial libraries, Cantley's group (23) demonstrated that peptides containing Leu at the –5 position, an aliphatic residue at –4, a basic residue at –3, and a hydrophobic amino acid at the +1 position (P-Ser site = 0) constitute optimal substrates for mammalian PKDs. Each critical position in the motif is occupied by an appropriate amino acid in Syntide-2 (PLAR TLSVAGLPGKK). A distinct peptide designed by Cantley's (23) rules for optimal substrates, AALVRQMSVAFFF, is also a good phospho-acceptor for DKF-1-mediated catalysis (Fig. 3A). Similarities among preferred substrates for DKF-1 and PKDs support the idea that the newly discovered DAG/PS-stimulated kinase is a new member of the PKD family. Steady-state kinetic analysis of DKF-1-catalyzed phosphorylation yielded apparent Km values of 11.7 ± 1.2 µM and 45.3 ± 3.5 µM for Syntide-2 and MgATP, respectively (Fig. 3B).
DKF-1 and PKD Are Activated by Different Mechanisms in Intact Cells—Activation of intracellular PKD by hormones, GFs, phorbol esters or second messengers is blocked by permeable PKC inhibitors (19–21, 26). Because purified mammalian PKDs are not markedly affected by the inhibitory drugs in in vitro kinase assays (75), these observations indicate that upstream PKCs regulate PKD activity and function. As expected, incubation of cells with a PKC inhibitor (GF109203X or RO31-8220) before and during treatment with PMA potently suppressed endogenous PKD activation (70–75% decline, Fig. 3C, right). In contrast, robust stimulation of DKF-1 caused by PMA was not altered by treating cells with a high concentration of inhibitor (Fig. 3C, left). PMA-mediated stimulation of PKCs, PKDs, and DKF-1 was abolished by 1 µM staurosporine, a nonselective inhibitor of a broad spectrum of protein kinases. Thus, suppression of mammalian PKD by GF109203X (or RO31-8220, data not shown) and the striking insensitivity of DKF-1 to these inhibitors (Fig. 3C) are not due to differences in accessibility of the kinase in situ. Instead, the data reveal inherent, fundamental differences between properties and modes of physiological regulation of the two PKD family isoforms. In vitro, GF109203X has no effect on DKF-1 kinase activity and (as expected) is only a weak, direct inhibitor of PKD (Fig. 3D). Results presented in Fig. 3, C and D, and previous studies (19–21, 26, 75) show that PKD activity is controlled by upstream PKCs. PMA stimulates DKF-1 kinase activity when PKCs in the same intracellular environment are inactive.
Bombesin Elicits PKC-independent Activation of DKF-1—The preceding results suggest that physiological activators of DAG synthesis will promote DKF-1 activation in situ. This proposition was tested in cells expressing the bombesin BB2 receptor. Bombesin receptors are present in many tissues and regulate a broad spectrum of physiological processes, including smooth muscle contraction, pancreatic secretion, gastrin release, satiety, lung development, thyrotropin secretion, lymphocyte chemotaxis, and activation of mammalian PKDs (28, 75–77). Bombesin binding with BB2, a serpentine (seven transmembrane segments) receptor, causes efficient and selective activation of heterotrimeric Gq/G11 GTP-binding proteins. G
q-GTP (or G11-GTP) binds and activates PLC, thereby stimulating DAG synthesis at the plasma membrane. Typically, accumulation of DAG is rapid but transient, because the second messenger is robustly metabolized.
Incubation of cells with 100 nM bombesin peptide increased DKF-1 kinase activity 9-fold within 2 min (Fig. 4A). Kinase activity declined 50% over the succeeding 18 min. A basal level of phosphotransferase activity was fully restored after 150 min (data not shown). The observations are consistent with (a) a rapid but transient elevation in DAG and (b) an ability of members of the PKD family to remain active for an extended time after DAG levels decline. Bombesin-induced translocation of DKF-1 to membranes is documented in a companion article (89).
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70% (Fig. 4B). Thus, bombesin and bombesin receptors trigger PLC/DAG-mediated, PKC-independent activation of DKF-1. Thr588 Regulates DKF-1 Catalytic Activity—Activation loops in mammalian PKD isoforms contain the sequence SFRRSV (amino acids 744–749 in PKD). Hormones or GFs that increase DAG content in membranes promote phosphorylation of both Ser744 and Ser748 (29, 30). Moreover, incorporation of phosphate at both target sites is essential for generating maximal phosphotransferase activity in the adjacent catalytic loop (amino acids 710–720 in PKD). In contrast, only Thr588 can be phosphorylated in the corresponding region of the predicted DKF-1 activation loop (QFRKTV, residues 584–589). This raised the possibility that DKF-1 and PKDs are activated by different mechanisms. Mutants were designed to test the importance of Thr588 in kinase activation; replacement of Thr588 with Ala will eliminate interactions mediated by the polar hydroxyl group and extinguish phosphorylation at the putative target site, whereas substitution of Thr588 with Ser might sustain normal function and regulation. Mutation of Thr588 to Ala sharply suppressed (>85%) basal and phorbol ester-stimulated DKF-1 phosphotransferase activity in situ (Fig. 5B, compare lanes 2 and 3 with lanes 7 and 8), whereas interchange of Ser for Thr588 produced a PMA-activated kinase that was indistinguishable from WT DKF-1 (Fig. 5B, compare lanes 2 and 3 with lanes 11 and 12). The results demonstrate the central importance of Thr588 in elevating and controlling DKF-1 kinase activity. Both WT DKF-1 and the Ala588 mutant were targeted to membranes upon incubation of cells with PMA for 10 min (Fig. 5C, and immunolocalization data shown in an accompanying article (89)).
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3-fold relative to WT DKF-1 (Fig. 5B, lanes 2 and 9). Incubation of transfected cells with PMA caused a large increase in specific kinase activity of WT DKF-1 (Fig. 5B, lanes 2 and 3). In contrast, phorbol ester treatment did not alter DKF-1(Glu588) activity (Fig. 5B, lanes 9 and 10). Like DKF-1(Glu588), DKF-1(Asp588) exhibits high basal activity that is not further enhanced by PMA.5 Replacement of Thr588 with nonacidic amino acids (e.g. Ala) yielded DKF-1 mutants that exhibit minimal basal activity and are unresponsive to PMA (Fig. 5B, lanes 7 and 8).
Possible linkage between phosphorylation of DKF-1 and expression of maximal DKF-1 catalytic activity with an optimal exogenous substrate was explored via a dephosphorylation strategy. PMA-activated DKF-1 was immunoprecipitated from cell extracts and incubated with highly purified protein phosphatase 2C (PP2C). Treatment with the phosphatase diminished DKF-1 phosphotransferase activity in a time- and temperature-dependent manner (Fig. 6). Under optimal conditions 90% of the kinase activity was abolished by PP2C.
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30–35% of the values determined for WT DKF-1 after exposure to saturating amounts of PMA. Despite this minor caveat, we conclude that the presence, precise context, and phosphorylation of a Thr or Ser hydroxyl group in the activation loop is evidently a stringent requirement for regulation of DKF-1 by PMA or DAG second messenger. Replacement of Gln584 with Ser yielded a DKF-1 activation loop sequence (SFRKTV) that closely resembled the motif containing two regulatory trans-phosphorylation sites in PKDs (SFRRSV). However, DKF-1 (Ser584) was activated with the same time course and reached the same maximal activity as the WT enzyme (data not shown). Moreover, WT and DKF-1(Ser584) activities were not inhibited by GF109203X or other PKC inhibitors (Fig. 5B, lanes 4–6). Thus, DKF-1 and PKDs are regulated by distinct upstream activators and different mechanisms.
Substitution of Thr588 with Ser did not affect properties of DKF-1. Both wild type and variant (DKF-1(Ser588)) kinases were stimulated 8-fold in cells incubated with PMA (Fig. 5B, lanes 2, 3, 11, and 12). Preincubation of cells with 3.5 µM GF109203X did not alter the magnitude of activation of DKF-1 or DKF-1(Ser588) by subsequent addition of PMA. Thus, the presence of Thr or Ser at amino acid 588 in the activation loop (QFRK(T/S)V) enables PKC-independent activation of DKF-1.
dkf-1 Gene Promoter Activity and Cognate DKF-1 Protein Kinase Are Selectively Expressed in a Subset of Cells in Vivo—Promoter/enhancer DNA that precedes the authentic dkf-1 structural gene was coupled to the 5' end of full-length DKF-1 cDNA. The resulting dkf-1P::DKF-1 minigene was inserted upstream from GFP reporter cDNA in a C. elegans expression plasmid. Subsequently, the dkf-1P::DKF-1·GFP fusion gene was integrated into C. elegans genomic DNA, and stable transgenic lines of C. elegans were propagated. Levels of DKF-1·GFP were monitored by Western immunoblotting (Fig. 7A), and expression of DKF-1 fusion protein in individual cells in vivo was assessed by fluorescence microscopy (Fig. 7, B–D). A highly reproducible pattern of DKF-1 expression was observed as animals matured from embryo to adult. Intense fluorescence signals corresponding to DKF-1·GFP revealed robust kinase accumulation in both (a) a region bounded by the anterior and posterior bulbs of the pharynx (Fig. 7, B, B2, and C) and (b) a tail area that contains lumbar, dorsorectal and pre-anal ganglia (Fig. 7D).
Specifically, DKF-1 is differentially enriched in a cluster of cells that are immediately adjacent to the posterior pharyngeal bulb (Fig. 7B). Strong signals also emanate from cells positioned along the lateral surface of this bulb in animals carrying the dkf-1P::DKF-1·GFP transgene (Fig. 7B2, arrows). At the anterior pharyngeal bulb, DKF-1 accumulates selectively in bodies and in very thin processes (dendrites and axons) of two neurons (Fig. 7C). High cell density and complex organization of neuronal ganglia in the head (and tail) region of C. elegans preclude determination of the exact identities of individual cells that exhibit elevated dkf-1 promoter activity and, therefore, enrichment in DKF-1·GFP polypeptide.
Despite the limitations listed above, a comparison of our current results with the C. elegans anatomy data base is quite informative. This approach focuses attention on a small group of "candidate DKF-1 positive cells" that will guide further studies. Nearly all cells expressing DKF-1 in Fig. 7, B, B2, C, and D, appear to be neurons. Two fluorescent cells with similar sizes and locations (at the anterior edge of the isthmusposterior bulb, Fig. 7, BN and B, arrows) may be M2 motor neurons. M2 neurons innervate muscles in the anterior bulb of the pharynx. The location of the more posterior fluorescent neuron in Fig. 7C (upper arrow) approximates the position of the cell body of an NSM neuron. NSM is a multifunctional neuron involved in sensing food and physical stretch, locomotory behavior, and secretion of growth factors and hormones. DKF-1 also accumulates in a cell resembling I1 (Fig. 7C, lower arrow), an interneuron that links sensory and effector neurons. Other candidate DKF-1-enriched cells in the pharyngeal region include: the AWB, ADL, and ADF chemosensory neurons; and AVB and AIA interneurons, which process sensory inputs and determine (directly or indirectly) the course of motor neuron actions at neuromuscular junctions, thereby governing body movement.
In C. elegans tail, DKF-1·GFP expression is differentially elevated in neurons located within the dense neuropile of several tail ganglia (Fig. 7D). This arrangement suggests that activation of the DKF-1 might generate signals involved in tail muscle contraction (movement). The pattern of fluorescence depicted in Fig. 7D reveals that cell bodies and/or processes of phasmid neurons (PHA, PHB, and PHC), interneurons (PVC, DVA, DVB, PVQ, PVT) and motor neurons (VD13, DD6, VA12) are candidate sites for accumulation of DKF-1 protein. The phasmid neurons have openings in the tip of the tail that receive chemosensory information from the environment. Sensory signals are routed to the pre-anal ganglion via phasmid processes that pass through lumbar commissures and synapse with various interneurons, including DVA, PVQ, AVA, and PVC, as well as motor neurons (VA12, DA9 etc.). Because some of the interneurons run along the nerve cords and terminate in the"brain"of the worm (nerve ring around the pharynx), it is possible that DKF-1 phosphotransferase activity plays an important dual role in routing local sensory data to both distal and proximal sites of integration (e.g. pharyngeal nerve ring and pre-anal ganglion, respectively). In concert with other inputs, DKF-1 activation would promote an appropriate, fully coordinated pattern of motor neuron activation/inactivation and muscle contraction/relaxation in response to positive or negative (e.g. attractants, toxins) environmental stimuli. This idea is supported by experimental data derived from"knock-out"animals carrying two disrupted copies of the dkf-1 gene (see Fig. 8 and under"Results"below).
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Depletion or Elimination of DKF-1 Protein in Vivo Reveals a Novel Physiological Role for a Member of the PKD Family—DKF-1 mRNA and protein levels were diminished in vivo by using RNAi methodology. Full-length DKF-1 cDNA was cloned into an RNAi vector (pPD139.06). The cDNA insert is located between opposing, IPTG-inducible, T7 promoters on sense and antisense DNA strands. E. coli was transformed with recombinant plasmid and double-stranded RNA (dsRNA) production was induced by adding IPTG. C. elegans cells take up dsRNA from ingested, transformed E. coli. Ubiquitous dsRNA transporters disseminate"silencing"reagent (RNAi) throughout the tissues of C. elegans (79, 80). Cells in the nematodes contain enzymes that cleave high molecular weight dsRNAs into small fragments (25 bp) that efficiently and selectively mediate degradation of a target transcript (DKF-1 mRNA in this instance) (81, 82).
L4 stage worms were fed with bacteria producing RNAi directed against DKF-1, and phenotypes of these worms and their progeny were observed with a dissecting microscope. DKF-1 protein content decreased dramatically (90%) in vivo when cognate RNAi was ingested (Fig. 8A, upper panel). In contrast, the concentration of a control protein (PKC-1) was not altered (Fig. 8A, lower panel). A distinctive phenotype was discovered in DKF-1-deficient C. elegans. Normal C. elegans traverse the agar medium on culture plates with a sinusoidal motion. This pattern is compromised by DKF-1 RNAi; worms exhibited a"zigzag"motility pattern with markedly altered amplitude (Compare Fig. 8B, panel 1 with panels 2 and 3). Two days after RNAi feeding, the mobility of adult worms was impaired. Severe or complete loss of muscle contraction near the anus caused the partially paralyzed tail region of the animals to be dragged along by the upper body. Thus, DKF-1 deficiency apparently causes a neuromuscular defect (uncoordinated (unc) phenotype) in the tail region.
A C. elegans gene disruption project in the laboratory of Judith Kimble (Department of Genetics, University of Wisconsin) facilitated the isolation of animals with a disrupted dkf-1 gene (generously provided by Liaoteng Wang, University of Wisconsin). Back-crossing with wild type C. elegans eliminated extraneous mutations and yielded viable worms with a dkf-1(–/–) genotype. Fragments of the disrupted dkf-1 gene were generated by PCR amplification using genomic DNA templates from individual worms in concert with a set of nested primers (Fig. 8C). Sequencing of PCR-amplified DNA revealed that a 1366-bp segment was deleted from the dkf-1 gene. This deletion corresponds to nucleotides 12736–14281 in the sequence of cosmid W09C5. Comparison of cosmid (genomic) and cDNA sequences indicates that exon 3 and portions of two introns are deleted from the dkf-1 gene. The expected splicing of exon 2 to exon 4 in the disrupted gene will cause a frameshift in DKF-1 mRNA that places a translation stop codon after amino acid 83. The resulting transcript will encode a chimeric protein composed of amino acids 1–78 of DKF-1 plus five C-terminal amino acids from an incorrect reading frame. The calculated Mr for the chimera is 9,600. Because the truncated DKF-1 polypeptide terminates before the first Cys-rich domain (C1a) and also lacks a kinase domain, the animals have a null phenotype for all established functional domains in DKF-1. dkf-1(–/–) C. elegans and WT worms that contain DKF-1 RNAi have the same"uncoordinated"phenotype (Fig. 8B, panels 3 and 2, respectively). Null mutants move normally from the time of hatching to the egg-laying stage and have no obvious defects in embryogenesis. Adult dkf-1(–/–) C. elegans display uncoordinated tail movement at day 5 (2 days after the final larval stage, L4) of the life cycle. The phenotype persists until death. A link between DKF-1 protein and null phenotype (unc) was rigorously confirmed by demonstrating that introduction of a dkf-1P::DKF-1·GFP fusion gene into dkf-1(–/–) animals corrected the movement disorder. Cell-specific expression of the transgene restored normal sinusoidal motion (data not shown). Western immunoblots demonstrated that the predicted 107-kDa DKF-1·GFP fusion protein accumulates in rescued transgenic nematodes. Endogenous 81-kDa DKF-1 is evident in extracts derived from wild type animals but is absent in samples prepared from C. elegans carrying two copies of the disrupted dkf-1 gene (Fig. 8D).
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