extfieldsofstudy: Medicine Biology

text:
Identification and disruption of an Arabidopsis zinc finger gene controlling seed germination

We describe here the Arabidopsis gene DAG1 , encoding a zinc finger transcription factor of the Dof family, and show that it is involved in the control of seed germination. By a reverse genetics approach, we isolated an Arabidopsis mutant line with one T-DNA insertion in DAG1 . Seeds from homozygous knockout dag1-1 plants do not develop dormancy and germinate also in the absence of light. Segregation analysis indicates that the effect of the mutation is maternal. Accordingly, in situ mRNA hybridizations reveal expression of DAG1 in the vascular tissue of the flower and maturing fruit but not in the seed. of several not after the the are

The seeds of several plant species do not germinate immediately after detachment from the mother plant, because the embryos that they contain are dormant. Dormancy, which is initiated during the final stages of seed development, is a trait of considerable adaptive significance-it prevents seeds from germinating under unfavorable conditions-as well as of great agricultural and biotechnological interest. A considerable body of evidence indicates that seed dormancy is induced by the plant hormone abscisic acid (ABA), which is also capable of inhibiting seed germination; mutants impaired in either ABA biosynthesis (aba) or sensitivity (abi) show reduced dormancy (Koornneef and Karssen 1994;Bewley 1997). Another class of plant growth factors, gibberellins (GA), are necessary for germination, and severe mutant alleles of the ga1 locus, which are required for GA biosynthesis, fail to germinate (Koornneef and Karssen 1994). Dormancy can be relieved by a period of dry storage of the seeds, and in some plant species, including Arabidopsis, germination requires light (Koornneef and Karssen 1994;Bewley 1997). However, the molecular mechanisms controlling seed dormancy and germination are, as yet, very poorly understood. Here, we report the identification of the Arabidopsis gene DAG1 (Dof affecting germination), encoding a Dof zinc finger transcription factor, and show that is involved in the control of seed dormancy.
The Dof proteins are a recently discovered family of plant transcription factors, ubiquitously present in the plant kingdom but absent from yeast and animals. These proteins are characterized by a strikingly conserved 52amino-acid segment, denominated Dof domain, encompassing a single CX 2 CX 21 CX 2 C zinc finger (Yanagisawa 1995;Zhang et al. 1995;De Paolis et al. 1996;Vicente-Carbajosa et al. 1997;Mena et al. 1998); in contrast, their sequences diverge widely outside the Dof domain. These features suggested that the Dof proteins are involved in the regulation of different functions typical of and of general relevance for plants. The few Dof proteins studied so far have been shown to be involved in gene regulation by light in maize (Yanagisawa and Sheen 1998), in the activation of storage protein genes in maize and barley (Vicente-Carbajosa et al. 1997;Mena et al. 1998), and in the the regulation of stress-related genes in Arabidopsis (Chen et al. 1996). Very recently, we have shown that the tobacco Dof protein NtBBF1 controls expression of the oncogene rolB in plants (Baumann et al. 1999).
We identified the Arabidopsis Dof gene DAG1 by sequence homology with the Dof domain of NtBBF1, and we isolated a dag1 knockout mutant by reverse genetics. The seeds of this mutant are nondormant and capable of germinating in the absence of light. This phenotype and its segregation pattern, as well as the pattern of expression of the gene, point to DAG1 as a gene that plays a novel and unexpected maternal role in controlling seed germination.

Isolation and structure of DAG1
An incomplete Arabidopsis DAG1 cDNA isolated previously by sequence homology with the Dof domain of the tobacco gene NtBBF1 (De Paolis et al. 1996) was utilized to isolate the corresponding genomic clone from an Arabidopsis library. Southern blot analysis indicated that the DAG1 protein is encoded by a single gene (not shown). The DAG1 gene was then mapped on chromosome 3 by screening of a YAC library (Camilleri et al. 1998) and localized between markers m424 and nga6. Analysis of the genomic sequence (EMBL accession no. AJ224122) revealed that DAG1 has two introns and encodes a 295-amino-acid putative protein with the single Dof zinc finger domain between amino acid positions 76 and 104.
∼22,000 independent tagged Arabidopsis lines of the Wassilewskija (WS) ecotype, led to the identification of one line with a T-DNA insertion in the DAG1 gene and subsequently to the isolation of the homozygous mutant dag1-1. The mutant was backcrossed with the wild type, and all subsequent characterizations were carried out on the F 2 homozygous progeny. Segregation of the kanamycin resistance marker associated with the T-DNA indicated the presence of a single insertion in DAG1, which was confirmed by Southern blot analysis (not shown). Sequencing revealed that the T-DNA is inserted close to the 3Ј splice site of the first intron of the DAG1 gene, as schematized in Figure 1. No DAG1 mRNA is detectable by either Northern or RT-PCR analysis in homozygous dag1-1 plants (not shown).

Phenotypic analysis
Development and growth of the dag1-1 knockout plants appear normal, and throughout the rosette (vegetative) stage mutants are indistinguishable from wild-type WS plants. Flowering dag1-1 plants are less branched than WS, but the number of inflorescences per branch is substantially the same and the length of the floral stalk is reduced only modestly (in average, 28.5 ± 0.7 cm for dag1-1 as compared to 32.8 ± 0.7 cm for WS plants). Flowering time and flower morphology of mutant and WS plants do not show appreciable differences.
Remarkably, however, all of the siliques (fruits) from dag1-1 plants have a characteristically twisted appearance, as shown in Figure 2, and are shorter than normal WS siliques (10.8 ± 0.2 mm vs. 13.1 ± 0.3 mm). Kanamycin-resistant heterozygous plants segregated the twisted silique phenotype as a recessive trait with a 3:1 ratio and plants with aberrant siliques proved to be homozygous for the T-DNA insertion, indicating that the mutant phenotype is linked to the insertion.

dag1-1 seeds are nondormant and do not need light to germinate
The morphological modification of the siliques observed in the dag1-1 mutant prompted us to check whether disruption of the DAG1 gene also affects seed development and/or viability. The twisted siliques were shown to carry a somewhat reduced number of seeds-on average 47.7 ± 4.5 seeds each, as compared to 58.0 ± 5.0 seeds in WS siliques-of normal appearance and containing morphologically normal embryos.
Disruption of the DAG1 gene has, in contrast, strong consequences on the germination of the seeds. As shown in Figure 3A, due to the effect of dormancy the rate of germination of freshly harvested WS seeds is rather low. In contrast, germination of dag1-1 mutant seeds reaches >95%, indicating that they are substantially nondormant. Because storage gradually releases Arabidopsis seeds from dormancy, germination tests were repeated 4 weeks after seed harvest. The capability to germinate normal seeds is, as expected, strongly enhanced by storage, whereas that of dag1-1 mutant seeds, being almost maximal upon harvest, is only fractionally affected (Fig.  3B).
Seed dormancy is induced by ABA, which also inhibits seed germination. Disruption of the DAG1 gene does not appear to affect sensitivity of the seeds to the hormone, as germination of both WS and dag1-1 knockout seeds is inhibited by the same concentration of ABA, as shown in Figure 3C.   Like WS seeds, dag1-1 seeds require GA to germinate: Figure 3D shows that germination of dag1-1 seeds is as sensitive as that of WS seeds to 10 µM paclobutrazol, an inhibitor of GA biosynthesis.
Most strikingly, although stored seeds from WS plants are absolutely incapable of germinating in the absence of light, those from dag1-1 mutant plants germinate in total darkness almost as well as in the presence of light (Fig. 3E). For normal Arabidopsis seeds to germinate, a pulse of red light is necessary to convert the (inactive) Pr form of the light receptor phytochrome to the active Pfr form (which is inactivated by far red light). We assessed that this is indeed the case for the WS ecotype seeds utilized in this work, as their negligible dark germination rate increased to 80% when WS seeds were exposed to a pulse of red light before performing the dark germination tests (Fig. 3F). The high germination rate of dag1-1 seeds in the dark is stimulated further by this initial pulse of red light and reaches 100%. We then tested whether the capability of mutant dag1-1 seeds to germinate in the absence of light is mediated by active phytochrome, by exposing the seeds to a Pfr-inactivating pulse of far red light prior to incubation in the dark. Figure 3F shows that Pfr is required for both the WS and the mutant seeds to germinate in the dark, as no germination of either was observed under these conditions.
The dag1-1 phenotype is due to disruption of DAG1 Conclusive evidence that the phenotype observed in dag1-1 plants is due to disruption of the DAG1 gene was obtained by transforming mutant plants with a genomic clone containing a wild-type copy of DAG1. F 3 transformants homozygous for the DAG1 transgene-identified through segregation of hygromycin-resistance trait conferred by the cloning vector-all showed normal-appearing siliques, indicating that DAG1 complements the twisted phenotype. The seeds from these siliques germinated in total darkness at the same low level of freshly harvested wild-type WS seeds, indicating that the exogenous DAG1 complements also the phenotype of dag1-1 seeds.

The effect of the disruption of DAG1 is maternal
To determine whether the phenotype of dag1-1 seeds is due to the mother plant or to the developing embryo, we analyzed the germination of stored seeds from heterozygous DAG1/dag1-1 plants. Two hundred seeds from DAG1/dag1-1 plants were sown in triplicate and scored for germination after 7 days in total darkness. The results are consistent with a maternal effect of this mutation, as none of the seeds from heterozygous DAG1/ dag1-1 plants is capable of germinating in the absence of light. Instead, up to one-fourth (dag1-1 homozygous) of the total seeds from heterozygous mothers would be expected to germinate in the dark, if the mutant phenotype were embryo-determined.

DAG1 is expressed in the mother plant but not in the embryo
In view of the maternal effect of the mutation and because seed dormancy initiates during embryo development, we analyzed expression of the DAG1 gene at different stages of development of Arabidopsis flowers, fruits, and embryos. The RT-PCR analysis shown in Figure 4A revealed that the DAG1 gene is expressed in flowers, as indicated by the amplification bands from flowers at stages 12 (lane F12) and 13 (lane F13), that is, before and, respectively, after flower opening (Smyth et al. 1990). After fertilization, DAG1 is expressed in developing siliques (stage 16, lane Si16) but not in fully mature ones (stage 17, lane Si17). As also shown in Figure 4A, in dag1-1 mutant plants no expression of the DAG1 gene is detectable in the corresponding developmental stages and organs. Control amplifications of the cDNA of ATL18 ribosomal protein from the same samples were all positive (not shown).
A more detailed view of the pattern of expression of DAG1 was obtained by in situ mRNA hybridizations. As shown in the longitudinal section of a stage 12 flower reported in Figure 4B, panel F12, a clear signal of DAG1 mRNA is visible in the gynoecium, specifically localized in the vascular tissue. Transverse sections of the gynoecium (panel Gy12) confirmed that the DAG1 mRNA signal is localized in the vasculature. After fertilization, expression of DAG1 in stage 16 developing siliques (panel Si16) also is clearly localized in the vascular bundles and extends in the funiculus that connects the placenta to the ovule, but no DAG1 mRNA signal can be detected in the ovule. In agreement with the RT-PCR data, no DAG1 mRNA signal is detectable in any tissue of stage 17 mature siliques (panel Si17). No DAG1 mRNA signal is visible at any stage of development in the embryo, as also shown in Figure 4, where sections of globular (panel GE) and torpedo (panel TE) embryos are reported. No DAG1-specific signal was detectable in any of the above sections with the DAG1 sense riboprobe (not shown). Thus, the fact that the pattern of expression of the DAG1 gene is limited to the mother plant is fully consistent with the maternal effect of the dag1-1 mutation.

Discussion
Relatively few Arabidopsis mutants with reduced or suppressed seed dormancy have been identified so far. In the seed maturation mutants of lec (leafy cotyledon) class lec1 (Meinke 1992) and fus3 (Bä umlein et al. 1994;Keith et al. 1994), dormancy is affected as a pleiotropic effect of mutations in (as yet unidentified) genes controlling embryo development. Nondormant lec1 and fus3 seeds contain morphologically abnormal embryos that develop into seedlings whose anatomical characteristics suggest a homeotic (Meinke 1992) and, respectively, heterochronic (Keith et al. 1994) interpretation of these two mutations. In contrast, embryos of the dag1-1 mutant appear normal and develop into normal seedlings.
Other seed dormancy mutants are affected in either ABA biosynthesis (aba, Koornneef et al. 1982;Lé on-Kloosterziel et al. 1996a) or ABA sensitivity (abi, Koornneef et al. 1984)-major evidence supporting the view that ABA is the main plant hormone responsible for the induction of dormancy. Of the abi mutants, only abi3 shows an effect limited to the embryo (Koornneef et al. 1984) and the ABI3 gene encodes a seed-specific transcription factor (Giraudat et al. 1992). ABI1 and ABI2 encode protein phosphatases 2C (Leung et. al. 1994(Leung et. al. , 1997Meyer et al. 1994), and their mutation alters several responses of the plant to ABA (Koornneef et al. 1984), as is the case for aba mutants (Koornneef et al. 1982;Lé on-Kloosterziel et al. 1996a). In contrast, disruption of DAG1 does not affect sensitivity of the seeds to ABA-germination of dag1-1 seeds is as sensitive to the hormone as that of WS seeds-and does not lead to the pleiotropic effects on water relations in adult plants characterictic of ABA-deficient mutants. In addition, although aba seeds do not require de novo synthesis of GA to germinate, dag1-1 seeds do: Like WS seeds, dag1-1 seeds are incapable of germinating in the presence of a concentration (10 µM) of paclobutrazol that inhibits GA biosynthesis (Lé on- Kloosterziel et al. 1996a). The reduced dormancy mutants rdo1 and rdo2, whose genes have not been isolated yet, are not affected in their response to ABA. In this respect, they are similar to the dag1-1 mutant, but their reduced dormancy trait is embryo-determined (Lé on- Kloosterziel et al. 1996b). In contrast, both the segregation pattern of the mutant phenotype in seeds from heterozygous plants and the lack of expression of the gene in the seeds indicate that DAG1 exerts its role from the mother plant.
To our knowledge, DAG1 is the first gene encoding a transcription factor that is specifically involved in the maternal control of seed germination. As yet, the only example of maternally determined loss of seed dormancy is in the mutants of the testa-the seed coat, which is of maternal origin. However, the enhanced germination of most of the testa mutants is due to altered physicochemical and/or mechanical properties of their seed coatevidence that the seed coat acts in preventing the embryonic radicle from protruding from the seed prematurely (Koornneef 1981(Koornneef , 1990Lé on-Kloosterziel 1997). Although testa mutations result in altered coloration, shape, or composition of the seed coat, dag1-1 seeds appear normal, suggesting that the maternal effect of the disruption of DAG1 does not involve the seed testa.
A most interesting trait of the nondormant dag1-1 mutant seeds is their capability to germinate in total darkness. In contrast, nondormant WS seeds (i.e., after storage-induced release from dormancy) still require (red) light to germinate. Red light is needed to turn the inactive Pr form of the phytochrome into the active Pfr form, which then triggers germination via a pathway, as yet totally unknown. The lack of light requirement of dag1-1 seeds, however, does not imply that they bypass the phytochrome-mediated pathway to initiate germination. When Pfr is inactivated by a pulse of far-red light, dag1-1 seeds fail to germinate in the dark.
On the other hand, DAG1 is expressed in the vascular tissue of the mother plant, suggesting that its mode of action involves transport to the seed of some component necessary for the control of dormancy. This evidence can be rationalized by a model whereupon DAG1 normally would ensure the inactivity (for dormancy initiation) of a (protein) component-transported from the mother plant to the seed-that would be activated (to trigger germination) as a consequence of red light perception. In plants where DAG1 is disrupted, this component would be in Cold Spring Harbor Laboratory Press on March 20, 2020 -Published by genesdev.cshlp.org Downloaded from the active form independent of red light: dormancy would not be established and seeds would germinate in the absence of light but germination could still be repressed by far-red light.
Further work is needed to verify this model or test alternatives. However, the identification of the first regulatory protein involved in the maternal control of seed germination opens a new avenue of research in this important and still largely obscure aspect of plant adaptation.

Materials and methods
Plant material and seed germination Seeds from Arabidopsis thaliana Heyhn, ecotype WS, were surface-sterilized as described by Santoni et al. (1994), sown into petri dishes on a solid medium containing Murashige and Skoog salts (Sigma), vitamins (Sigma), 0.8% agar, and 1% sucrose, and kept in the dark at 4°C for 2 days. The dishes were then transferred into the growth chamber (24°C/ 21°C, 16/8 hr day/night, 300/µEinstein/m 2 ). Seedlings were transferred into individual pots 8 days after germination, and maintained in the growth chamber. Thirty WS and dag1-1 flowering plants were analyzed for morphometric parameters, and 30 siliques from both types of plants were measured and scored for the number of seeds contained.
For germination experiments, surface-sterilized seeds were sown in triplicate in Petri dishes (30-60 per dish) over one layer of Whatmann paper saturated with 2.5 ml of sterile distilled water and kept in the dark at 4°C for 2 days. Germination tests were subsequently carried out under the illumination conditions utilized for plant growth or in total darkness. In the text, stored seeds are seeds stored for 4 weeks after harvest in the dark under dry conditions at room temperature. For ABA and paclobutrazol sensitivity tests, seeds were sown on 0.8% agar in the presence of the ABA and paclobutrazol concentrations indicated in Figure 3, C, and, D, respectively; germination was scored after 7 days of incubation under standard illumination conditions.
For light-pulse experiments, light sources were from Phylips (bulb), and fluence rates were as follows: red light, bulb TL (40 W), fluence, 316 µW/cm 2 ; far-red light, bulb Phylinea (60 W), fluence, 350 µW/cm 2 . Seeds were exposed to red or far-red light for 5 min and kept in the dark for 7 days before scoring for germination.

Molecular techniques
Molecular techniques were perfomed according to standard protocols. Arabidopsis DNA was extracted according to Bouchez et al. (1993). Total RNAs were extracted from wild-type and mutant plants as described in Vittorioso et al. (1998). The RT-PCR analysis was performed with DAG1-specific primers, according to the manufacturer's instructions (Perkin-Elmer).

Isolation of DAG1
The Dof domain of the previously isolated partial cDNA of DAG1 (De Paolis et al. 1996), was utilized for the screening of an Arabidopsis genomic library (Clontech). Several positive clones were isolated. An 8-kb SalI insert containing the entire DAG1 gene was cloned into pBluescript KS+ vector (Stratagene) for restriction analysis. The 1.5-and 3.0-kb EcoRI fragments containing the promoter (1 kb) and the DAG1 gene, respectively, were subcloned and sequenced. Full-length sequence of the cDNA was obtained by 5Ј-RACE, according to the manufacturer's instructions (GIBCO-BRL). DNA sequencing was performed with an ABI373A automated DNA sequencer as recommended by the manufacturer (Applied Biosystems). The analysis of DAG1 cDNA and derived protein sequence was performed using GCG and BLAST, or the National Center for Biotechnology Information (NCBI, Bethesda, MD).
Isolation of the dag1-1 knockout plant A collection of T-DNA insertion mutants (Laboratoire de Gé netique et Amé lioration des Plantes, INRA, Versailles, France) containing ∼22,000 individual lines, was screened by PCR utilizing pairs of primers corresponding to both the T-DNA right and left borders and the DAG1 3Јspecific region. One positive line was identified and isolated. The position of the T-DNA within the DAG1 gene was determined by sequencing the PCR-amplified fragment. The DAG1-tagged line was shown to contain a single T-DNA insert and checked for linkage of the T-DNA insert to the DAG1 gene. More than 100 putative heterozygous plants (kanamycin resistant with normal siliques) segregated 25% mutant progeny (twisted siliques). Plants heterozygous for the mutation were self-fertilized, and the transmission of the phenotype was confirmed in the F 3 generation.
Complementation of the dag1-1 mutant A 2.7-kb genomic fragment corresponding to the DAG1 gene was cloned in the pCAMBIA binary vector (Canberra, Australia). dag1-1 homozygous plants were transformed by in planta infiltration (Bechtold et al. 1993) and transformants selected on 40 µg/ml hygromycin. The F 3 progeny from two individual hygromycin-resistant plants was analyzed for complementation of the dag1-1 phenotypes.
In situ mRNA hybridizations A 400-bp PCR fragment containing the carboxy-terminus of the DAG1coding sequence up to the 3Ј UTR, was cloned in the pCR2.1 vector (Stratagene). For the antisense riboprobe, the construct was linearized and in vitro transcribed with T7 RNA polymerase in the presence of 3 H-labeled UTP. The sense probe was derived from a construct containing the same PCR-amplified fragment cloned in the opposite orientation. Conditions for tissue fixation, paraffin embedding, hybridization (on 8-mm sections), and washings were as described by Drews et al. (1991).


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Hydrophobic Residues Phe 751 and Leu 753 Are Essential for STAT5 Transcriptional Activity*

One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and acti-vators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation. Surpris-ingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe 751 or Leu 753 to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery. Cytokines initiate their pleiotropic effects on cells by binding to specific transmembrane receptor proteins. This association induces a conformational change in the receptor (1, 2) that signals to the nucleus via a direct

One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe 751 or Leu 753 to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery.
Cytokines initiate their pleiotropic effects on cells by binding to specific transmembrane receptor proteins. This association induces a conformational change in the receptor (1, 2) that signals to the nucleus via a direct pathway involving the activation of receptor-associated Janus tyrosine kinase (JAK), 1 and the subsequent phosphorylation and nuclear translocation of members of the signal transducer and activator of transcription (STAT) family (3,4). Once activated, JAK phosphorylates the receptor on cytoplasmic tyrosine residues providing sites for STAT proteins to bind to via their Src homology 2 (SH2) do-mains (5)(6)(7)(8). The juxtaposed JAK phosphorylates STAT on a tyrosine residue C-terminal to its SH2 domain (9 -12). Phosphorylated STAT dissociates from the receptor and homo-or heterodimerize through a reciprocal interaction between the phosphotyrosine of one STAT and the SH2 domain of the other STAT (13,14). These active dimers translocate to the nucleus, possibly through a nucleoprotein interactor-mediated process (15), where they bind specific DNA elements to activate transcription of target genes (16,17).
Seven mammalian STATs, ranging in size from ϳ90 to ϳ115 kDa, as well as several alternatively spliced variants have been documented (3,4). The most conserved feature about the STAT family is their SH2 domain (13,14). As already alluded to, this region is indispensable for activation and dimerization of the STAT proteins. N-terminal to this domain, but separated by a linker region, is the DNA-binding domain (17)(18)(19). The crystal structures for DNA-bound STAT-1 and -3 homodimers, lacking their C termini, have revealed that STAT dimers bind DNA in a saddle-like structure (18,19).
There are several lines of evidence to suggest that the Nterminal region of STATs is involved in protein-protein interaction. The N terminus of STAT-1 is required for the tyrosine dephosphorylation of STAT-1 and has been shown to regulate the nuclear translocation of and to mediate the interaction between STAT dimers (20 -23). Furthermore, the transcriptional co-activator, p300/CBP, can associate with the N terminus of STAT-1 (24). Recently, it was shown that Nmi can interact with the N-terminal coiled-coil domain of several STATs and enhance their function (25). In particular, association of Nmi with STAT1 and STAT5 facilitated the recruitment of p300/CBP to the complex and enhanced their transactivation following interferon-␥ and IL-2 stimulation, respectively.
The least conserved feature of STATs is their C termini. Alternatively spliced variants lacking the C-terminal region as well as mutants in which the C terminus was truncated has provided evidence that this region acts as the transactivation domain (11,26,27). In the case of STAT5, these mutants show sustained tyrosine phosphorylation and exhibit increased levels of DNA binding activity compared with the wild-type protein but remain transcriptionally inert (26,28). In addition, the C-terminal region of STATs has been described to interact with various co-activators, such as MCM5 (29) and p300/CBP (24, 30 -34).
These observations suggest that the C terminus of STATs has pleiotropic functions and plays an important regulatory role. To better understand the function of the C termini of STATs, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have identified a 12-amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using sitedirected mutagenesis, we show that, within this 12-amino acid region, acidic residues were non-essential for transactivation. In contrast, the non-acidic residues, especially the hydrophobic residues Phe 751 and Leu 753 , were crucial for transactivation.

EXPERIMENTAL PROCEDURES
Chemicals, cDNAs, and Antibodies-Recombinant murine IL-3 was purchased from R&D (Minneapolis, MN). Recombinant murine prolactin was obtained from the National Hormone and Pituitary Program (NIDDK, National Institutes of Health, Bethesda, MD). Staurosporine was obtained from Sigma, dissolved in dimethyl sulfoxide (Me 2 SO), and used at a final concentration of 500 nM. Murine HA-JAK2 and HA-STAT5a cDNAs were obtained from Alan D. D'Andrea (Dana-Farber Cancer Institute, Boston, MA). Murine prolactin receptor cDNA was generously provided by Bernd Groner (Institute for Experimental Cancer Research, Tumor Biology Center, Freiburg, Germany). An internal ribosome entry site/green fluorescent protein (IRES/GFP) cDNA was obtained from Richard Mulligan (Children's Hospital, Boston, MA). Anti-HA antibody (12CA5) was purchased from Roche Molecular Biochemicals. Mouse anti-phosphotyrosine-specific STAT5 monoclonal antibody (ST5P-4A9) was purchased from Zymed Laboratories Inc. (South San Francisco, CA).
cDNA Plasmids-The truncated STAT5 mutant (⌬737) was generated by introducing a stop codon at amino acid 737. A Y694S mutation in STAT5a was introduced into its cDNA by the polymerase chain reaction, generating a BamHI site. This BamHI site was used to subclone the 3Ј end of the STAT5a coding region (a 306-base pair BamHI/ EcoRI fragment) into pBluescript (pBS-CT-STAT5). All subsequent mutants were generated by polymerase chain reaction on this construct using the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All mutations were confirmed by sequencing and the NsiI/ EcoRI fragment subcloned back into the full-length, wild-type cDNA in the mammalian expression vector pcDNA3. To generate the GAL4-Cterminal STAT5 fusion constructs, the BamHI/EcoRI fragment of the various mutants in pBS-CT-STAT5 were subcloned into the BamHI/ EcoRI sites of the GAL4 DNA-binding domain fusion expression vector, pFA-CMV (Stratagene). All cDNAs were purified using Qiagen Maxiprep kits (Qiagen Inc., Valencia, CA).
COS-7 Transfections and Extracts-Confluent COS-7 cells were split one into five the day prior to transfection. Approximately 50% confluent plates were transiently transfected for 5 h at 37°C with 1 g of JAK2 and 5 g of STAT5 cDNAs per dish using LipofectAMINE/Opti-MEM (Life Technologies, Inc.) according to the manufacturer's directions. Cellular extracts were prepared at either 24 or 48 h after transfection. Cells were washed with phosphate-buffered saline, resuspended in lysis buffer (1% Nonidet P-40, 50 mM Tris.HCl, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 50 mM NaF, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 2 g/ml aprotinin C, and 0.5 g/ml leupeptin), and incubated on ice for 30 min. Extracts were centrifuged (4°C) for 10 min at 13,000 rpm, and the resulting supernatants were used for subsequent immunoprecipitations, direct immunoblotting, or electrophoretic mobility shift assay (EMSA). For some experiments, STAT5 EMSA was performed using nuclear extracts prepared as described previously (38).
Immunoprecipitations and Immunoblotting-Anti-HA antibody (1 g) was added to cellular extracts and incubated overnight at 4°C in the presence of protein A-Sepharose (Amersham Pharmacia Biotech). Immune complexes were washed thrice with Nonidet P-40 lysis buffer before addition of 2ϫ Laemmli sample buffer. Bound proteins were eluted by boiling for 10 min and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using 6 -7% gels. For direct immunoblotting, extracts (25-50 g) were mixed with 2ϫ sample buffer, boiled, and separated by SDS-PAGE.
Electrophoresed proteins were transferred to Immobilon-P PVDF membrane (Millipore, Bedford, MA) and blocked with 3% BSA in TBST (Tris-buffered saline plus 0.05% Tween 20). Anti-Tyr(P)-STAT5 (3 g) was diluted in 1% BSA/TBST and incubated for 2 h at room temperature. Anti-HA antibody (4 g) was added to blocking solution and incubated overnight at 4°C. Membranes were washed four times with TBST and incubated with a 1:5000 dilution of horseradish peroxidaseconjugated anti-mouse Ig (Amersham Pharmacia Biotech) in 1% BSA/ TBST for 30 min at room temperature. After four washes with TBST, immunoreactive proteins were detected using enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech). RC20 blotting was performed as described previously (35). Where appropriate, membranes were stripped with a solution containing 2% SDS, 62.5 mM Tris, and 0.7% ␤-mercaptoethanol for 30 min at 55°C, washed extensively with H 2 O, twice with TBST, and re-blocked with 3% BSA/TBST before addition of primary antibody.
Electrophoretic Mobility Shift Assay-Samples (5 g) of extracts were used for STAT5 EMSA. EMSA was performed with a STAT5 oligonucleotide probe from the ␤-casein promoter element (top strand: 5Ј-GTAGATTTCTAGGAATTCAAA-3Ј) as described previously (35). Association and dissociation of the STAT5-DNA complex was determined using modifications to an established protocol (39). Briefly, to measure association, 5 g of extracts were incubated at 0°C with labeled probe before quenching with a 100-fold excess of specific competitor. For dissociation experiments, a steady-state complex was initially achieved by pre-incubating 5 g of extracts with labeled probe for 30 min at 0°C, then a 100-fold excess of unlabeled probe was added before incubating at 30°C. Dissociation was dependent on the addition of unlabeled probe since poly(dI)⅐poly(dC), a nonspecific competitor DNA, failed to compete with the STAT5-DNA complex (data not shown). For both association and dissociation experiments, samples were immediately loaded at the indicated times onto continuously running 6% non-denaturing polyacrylamide, 0.5ϫ TBE gels. For more details, see legend to Fig. 6. All quantitation was done by phosphorimage analysis using a GS-525 molecular imager system (Bio-Rad).
STAT5 Transactivation Assays-Confluent NIH3T3/ProR cells were split one into three the day prior to transfection. Approximately 50% confluent plates were transiently transfected for 5 h at 37°C with 2 g of (STAT5RE) 6 -61pF* and 3 g of STAT5 cDNAs per dish using Lipo-fectAMINE/Opti-MEM. 100 ng of a Renilla luciferase construct was included to normalize for transfection efficiency. After 24 h, cultures were split and cultured in either the presence or absence of 10 g of recombinant murine prolactin for 16 -20 h. Cells were harvested and assayed for luciferase activity using a dual luciferase assay kit (Promega, Madison, WI). Activity was normalized with Renilla luciferase activities and expressed relative to that obtained with prolactin-stimulated wild-type STAT5 (100%).
For GAL4 fusion experiments, NIH3T3/ProR cells were prepared as described earlier before lipofection with 6 g of the GAL4-fusion cDNA, 4 g of the GAL4 reporter gene pFR-LUC (Stratagene), and 100 ng of Renilla luciferase construct. After 16 -20 h cells were harvested and luciferase activities determined. BaF 3 cells (5 ϫ 10 6 ) were prepared as described previously (36) before being electroporated at 350 V and 960 microfarads with 5 g of the GAL4-fusion cDNA, 7.5 g of pFR-LUC, and 100 ng of Renilla luciferase construct. After 16 -20 h cells were harvested and luciferase activities determined. Activity was normalized with Renilla luciferase activities and expressed as a percentage of GAL4-WT STAT5 activity.

RESULTS
Internal Deletions within the STAT5 C Terminus Abolish the Transcriptional Activity of GAL4 Chimerae-Truncation analysis of the C terminus of STAT5 had indicated that the region between amino acids 750 and 772 played an important role in transactivation (26). To further understand the contribution of this region to transactivation, we generated a series of Cterminal mutants of STAT5 (Fig. 1A). The C termini of these mutants, starting from residue 694, was fused to the GAL4 DNA-binding domain (DBD) (Fig. 1B) and the relative ability of these constructs to transactivate a GAL4-responsive luciferase reporter gene was examined by ectopically expressing these chimerae in BaF 3 cells. As shown in Fig. 2A, fusing the wildtype (WT) STAT5 C terminus to the GAL4-DBD resulted in luciferase expression 10 -20-fold higher than the GAL4-DBD alone. A C-terminal truncation of the last 57 amino acids (⌬737) removed all of STAT5's ability to transactivate confirming that the STAT5 transactivation domain is localized within its C terminus. Internal deletions of 29 (TD⌬29) and 12 (TD⌬12) amino acids also dramatically decreased the ability of these mutant chimerae to transactivate. However, unlike the truncated mutant, these internally deleted mutants, especially TD⌬12, were not completely inert. A potential SH3 binding site within the 29-amino acid region was destroyed by mutating four proline residues to alanine (P 4 /A 4 ). This mutation had no effect on the ability of the GAL4-chimera to transactivate compared with the WT fusion protein, suggesting the structural contributions of these proline residues were non-essential for transactivation. Identical results were obtained when we repeated these experiments in NIH3T3 cells (Fig. 2B). Since the P 4 /A 4 mutant had 100% WT activity in BaF 3 cells, we did not test this mutant in NIH3T3 cells.
Internal Deletions within the C Terminus Preserve Transactivation by STAT5 Mutants-To extend these observations, we placed these mutations in the context of the STAT5 backbone and we examined the relative ability of these constructs to transactivate a STAT5-responsive promoter in NIH3T3 cells stably expressing the prolactin receptor (NIH3T3/ProR cells). STAT5 constructs were co-transfected into these cells with a STAT5 luciferase reporter gene, and luciferase expression was measured following activation of the ectopically expressed STAT5 proteins in response to prolactin treatment. As shown in Fig. 3, expression of WT STAT5 resulted in a 4-fold increase in luciferase expression following prolactin treatment. This increase was specific as the Y694S mutant, which cannot be phosphorylated (and therefore cannot dimerize and bind DNA), failed to transactivate the reporter gene. Furthermore, the transfection of pcDNA3 failed to induce luciferase expression following prolactin treatment (data not shown), indicating that prolactin-induced luciferase expression was dependent on ectopically expressed STAT5. The P 4 /A 4 mutant also transactivated the reporter gene, although in this case to 70% of WT levels in response to prolactin treatment. Prolactin activation of the ⌬737 mutant failed to significantly increase luciferase expression above that of the untreated sample, confirming our earlier experiments that the C terminus contains the transactivation domain. Unexpectedly, we observed that both the TD⌬29 and TD⌬12 mutants transactivated the STAT5 reporter gene to 75% and 100% of WT levels following prolactin treatment, respectively. This was in contrast to our data using GAL4 fusion proteins, which clearly demonstrated that these deletions affect the activity of the transactivation domain per se (Fig. 2) and suggested that deletion of this region of the C terminus, in the context of the native protein, had additional effects on STAT5 function.
Internal Deletion of the STAT5 C Terminus Enhances DNA Binding Activity-In an effort to explain the discrepancy in the previous experiments, we expressed the C-terminal mutants in COS-7 cells and examined whether the mutations altered the function of STAT5 by affecting DNA binding or tyrosine phosphorylation. Co-expression of the ⌬737 mutant and JAK2 in COS-7 cells resulted in a marked increase in DNA binding activity compared with cells expressing WT STAT5 (Fig. 4A). Transfection of either WT STAT5 or JAK2 cDNAs alone did not result in detectable STAT5 DNA binding activity, indicating that the co-expression of both STAT5 and JAK2 is necessary for measurable STAT5 DNA binding activity in COS-7 cells (data not shown). Since STAT5 is normally phosphorylated on Tyr 694 upon activation (40), we confirmed that phosphorylation of Tyr 694 was essential for STAT5 DNA binding activity, as mutation of this residue to serine (Y694S) rendered the protein incapable of being tyrosine phosphorylated and abrogated DNA binding ( Fig. 4A; data not shown). We also examined the status of Tyr 694 in the ⌬737 mutant using a Tyr(P) 694 -specific-STAT5 antibody. We observed an increase in Tyr 694 phosphorylation, which largely explained the enhanced DNA binding activity seen with the ⌬737 mutant.
Next we examined whether the TD⌬29 and TD⌬12 mutations affected DNA binding or tyrosine phosphorylation. We observed a markedly enhanced level of DNA binding activity for both mutants (Fig. 4B). The increase was comparable to that observed with the ⌬737 mutant. As with ⌬737, this effect was largely due to an increase in Tyr 694 phosphorylation in both TD⌬29 and TD⌬12 mutants. Tyrosine 694 was essential for the enhanced DNA binding activity and tyrosine phosphorylation observed with these mutants, since mutation of this residue to serine (Y694S) abolished all phosphorylation and DNA binding activity in the double mutant ( Fig. 4B; data not shown). Although the 29-amino acid region loosely corresponded to a PEST sequence (41), deletion of this region did not affect the level of protein expression (Fig. 4B, lower panel), suggesting this region of STAT5 is not involved in protein stability. In the case of the P 4 /A 4 mutant, we observed a slight reduction in DNA binding activity compared with WT STAT5. This correlated well with the decrease in Tyr 694 phosphorylation in this mutant, suggesting that the prolines may be required for optimal phosphorylation of Tyr 694 by JAK. These results confirm that the internal C-terminal deletions of 29 and 12 amino acids mimicked the effects of the C-terminal truncation (⌬737 mutant). In addition, they demonstrated that the extreme C terminus of STAT5 is not essential for normal levels of DNA binding or Tyr 694 phosphorylation.
Internal Deletion of the C Terminus of STAT5 Delays Tyrosine Dephosphorylation-To determine the mechanism of the deletion mutant's enhanced DNA binding activity, we investigated the effects of the 29-amino acid deletion (TD⌬29) on tyrosine dephosphorylation of STAT5. We stably expressed the WT or TD⌬29 STAT5 proteins in BaF 3 cells and established clonal lines from single cells expressing high levels of the HA-tagged proteins. After stimulation with IL-3, the mutant protein was dephosphorylated at a slower rate compared with that of WT STAT5 (Fig. 5A). Interestingly, an associated phosphoprotein (pp59) was consistently observed to co-precipitate with HA-tagged STAT5 proteins expressed in BaF 3 cells. Its phosphorylation and association profile closely parallels that of either mutant or WT STAT5 phosphorylation. Although we have not yet identified pp59, our data suggest that it is unlikely to be a degradation product of STAT5 (data not shown). To further confirm that the 29-amino acid deletion affects the rate of dephosphorylation rather than that of phosphorylation, WT or TD⌬29 STAT5 cDNAs were co-transfected into COS-7 cells with JAK2. After 24 h, staurosporine was added to inhibit JAK2 activity required for phosphorylation of the ectopically expressed STAT5 proteins. Although WT STAT5 DNA binding activity could no longer be detected after 1 h of staurosporine treatment, the DNA binding activity of TD⌬29 was unaffected up to 4 h after treatment, indicating a slower rate of tyrosine dephosphorylation (Fig. 5B). Together, these experiments indicate that TD⌬29 is dephosphorylated on Tyr 694 more slowly than WT STAT5. Furthermore, the resulting increase in steady state STAT5 dimers due to prolonged phosphorylation partially explains the enhanced DNA binding activity of the mutant protein.
Careful examination of mutants which exhibit a markedly enhanced DNA binding characteristic (i.e. ⌬737, TD⌬29, TD⌬12) revealed that they do so in a manner that is disproportionately greater than their respective enhanced levels of Tyr 694 phosphorylation would predict (Fig. 4). This suggested that, in addition to altering the kinetics of dephosphorylation, these mutations were in some way affecting DNA binding, perhaps affecting their DNA binding affinity. To explore this possibility, we determined the respective "on" and "off" rates of DNA binding for TD⌬12 and WT STAT5 proteins. Surprisingly, neither the on or off rate of DNA binding was significantly affected by the 12 amino acid deletion (Fig. 6). Thus, it appears that the mutations do not affect DNA binding per se. We believe that the mutations favor the rate of dimerization once the mutant proteins are phosphorylated. This would effectively raise the concentration of DNA-binding complex relative to what is normally observed with WT STAT5.
Non-acidic Residues, but Not Acidic Residues, within the 12-Amino Acid Region Are Crucial for Transactivation by STAT5-To better understand how this 12-amino acid region affects STAT5 function, we made additional mutations within this region. The 12-amino acid domain, which is essential for transactivation, contains one serine (Ser 756 ). Since serine phosphorylation has been shown to be important for maximal transactivation by STATs (29,42), we mutated this residue to glycine (S756G; Fig. 7A). Additionally, since acidic residues have been reported to be important for transactivation (43)(44)(45)(46), we made two additional mutants in which we mutated either all of the acidic (TDacid/Ala) or all of the non-acidic (TDnon-acid/Ala) residues within this region to alanine residues (Fig. 7A).
Using the GAL4 chimera transactivation assay, we observed that the S756G mutation resulted in levels of transactivation 45% and 70% of WT when tested in BaF 3 and NIH3T3 cells, respectively (Fig. 7, B and C). The acidic mutant (TDacid/Ala), although inactive in BaF 3 cells, retained nearly 50% of WT activity when expressed in NIH3T3 cells. We favor the explanation that this difference reflects differential expression of the acidic mutant in the two cell lines. Indeed, it is the only mutant for which we see reduced levels of protein expression when expressed in COS-7 cells (see below), suggesting it is less stable than the WT or other mutant proteins.
When placed in context of the STAT5 backbone and expressed in fibroblasts, the S756G mutation resulted in 75% of WT levels of transactivation following prolactin treatment (Fig.  7D), similar to that observed in fibroblasts using the GAL4 chimera. Interestingly, despite weakening the transactivation domain in the GAL4 chimera, the TDacid/Ala mutation resulted in greater than WT levels of transactivation when placed in the STAT5 backbone, suggesting that this mutation might also affect DNA binding activity. Somewhat to our surprise, we found that the TDnon-acid/Ala mutation abolished transactivation either in the context of the GAL4 chimera or in the context of the STAT5 molecule (Fig. 7, B-D).

Loss of Acidic Residues within the 12-Amino Acid Region
Enhances DNA Binding Activity-Ectopic expression of the S756G mutant in COS-7 cells affected neither DNA binding activity nor Tyr 694 phosphorylation when compared with WT STAT5 (data not shown). In contrast, expression of the nonacidic mutant resulted in an increased level of DNA binding activity compared with WT STAT5. However, this increase was less dramatic than that observed with the TD⌬12 mutant (Fig.  8). Furthermore, it correlated very well with an increase in phosphorylation of Tyr 694 . Expression of the acidic mutant also resulted in enhanced DNA binding activity, similar to that observed with the non-acidic mutant. However, unlike the nonacidic mutant, the acidic mutant had significantly decreased levels of protein expression and Tyr 694 phosphorylation compared with WT STAT5 (Fig. 8). Despite its reduced expression and phosphorylation, the acidic mutant was still capable of binding more DNA than the WT protein, indicating that loss of the acidic residues within the 12-amino acid domain significantly augments DNA binding activity (possibly through an increased rate of dimer formation).
Hydrophobic Residues Phe 751 and Leu 753 Are Essential for STAT5 Transactivation-The loss of transcriptional activation associated with the TDnon-acid/Ala mutation led us to more closely investigate the role of hydrophobic residues within the 12-amino acid region. When we compared the minimal transactivation domain of the herpes simplex virus transcription factor, VP16, with our 12-amino acid region of STAT5, we found that there was a homologous sequence EFDLD common to both proteins (Fig. 9A). Phenylalanine and leucine residues in this region were shown to be critical for transactivation by VP16 (47,48). Interestingly, the same residues in STAT5 had been mutated as part of the non-acidic mutation that abolished transactivation. We therefore tested whether both Phe 751 and Leu 753 of STAT5 might also be critical for transactivation. We made three additional mutants in which Phe 751 or Leu 753 or both were mutated to alanine residues (Fig. 9B). Since the non-acidic mutant was transcriptionally inert using either a GAL4 chimera or the STAT5 backbone, we only tested the activity of these mutants using GAL4 chimerae in BaF 3 cells. As seen in Fig. 9C, mutating either Phe 751 or Leu 753 to alanine reduced transactivation to 25% and 30% of WT STAT5, respectively. The double mutant (F751A/L753A) did not result in any further decrease in transcriptional activity compared with the F751A mutation, suggesting that Phe 751 may be more essential than Leu 753 . These results indicate that the hydrophobic residues within the 12-amino acid region of STAT5, especially Phe 751 and Leu 753 , are crucial for transactivation.

DISCUSSION
Using deletion and truncation mutants, we have provided further evidence that the STAT5 transactivation domain is localized within its C terminus. Specifically, GAL4-chimerae have defined a crucial 12-amino acid region, which, when deleted, resulted in only residual levels of transactivation. Surprisingly, when we deleted these 12 amino acids from the entire STAT5 molecule, we found that transactivation by this mutant STAT5 was preserved. Biochemical analysis of this mutant in COS-7 cells revealed that deletion of these 12 amino acids resulted in a marked increase in DNA binding activity. Therefore, this 12-amino acid deletion has two effects. First, it weakens, but does not abolish all transcriptional activity, and second, it markedly enhances DNA binding activity. The combination of these two effects compensate for each other, resulting in a STAT5 mutant with net activity similar to the WT protein. Hence, the function of the C terminus of STAT5 is pleiotropic with somewhat "plastic" properties such that considerable mutation in this region can be tolerated before an overall loss of function is observed.
The enhanced DNA binding activity seen with several STAT5 mutants in this study is largely due to an increase in phosphorylation of Tyr 694 . This increase in DNA binding activity was critically dependent on Tyr 694 , since mutation of this residue to serine rendered the double mutant proteins incapable of being phosphorylated and binding DNA. We have shown previously that dephosphorylation of Tyr 694 is the most likely mechanism for the inactivation of STAT5 (35). In this regard, we found that the TD⌬29 mutant had a slower rate of tyrosine dephosphorylation. which is consistent with that seen with variants of STAT5 lacking the C terminus (28). Thus, a delay in dephosphorylation causes a slower inactivation of TD⌬29 and consequently a steady-state accumulation of active dimers leading to an increase in DNA binding activity. Furthermore, the DNA binding activity and Tyr 694 phosphorylation of both our truncated (⌬737) and deletion (TD⌬12) mutants are similarly affected. Hence, the extreme C terminus of STAT5 is not involved in the normal regulation of DNA binding or Tyr 694 phosphorylation; rather, the essential 12 amino acids are. Whether or not this domain recruits a phosphatase or merely modulates its activity remains to be determined.
We noted that an increase in Tyr 694 phosphorylation alone does not entirely account for the enhanced DNA binding characteristic for several of our mutants. The ⌬737, TD⌬29, and TD⌬12 mutants all bound more DNA than WT STAT5, yet they FIG. 6. Deletion of the essential 12-amino acid domain does not affect DNA binding affinity. COS-7 cells were co-transfected for 24 h with HA-JAK2 and either WT or TD⌬12 HA-STAT5a cDNAs. A, to measure dissociation of STAT5-DNA complex, a 100-fold excess of unlabeled probe was added to a pre-formed, steady-state complex (5 g of extracts) prior to incubating at 30°C for the indicated time. Gels were dried, quantitated, and the fraction bound at time zero was plotted against time. B, to measure association of STAT5-DNA complex, 5 g of extracts were incubated at 0°C with labeled probe as indicated before quenching with a 100-fold excess of unlabeled competitor. Gels were dried and quantitated, and the amount of bound probe (% max) was plotted against time. Solid squares, WT; open squares, TD⌬12. did so in a manner that was disproportionately greater than their respective levels of Tyr 694 phosphorylation would predict (Fig. 4). For example, the TD⌬12 mutant showed a 2-3-fold increase in Tyr 694 phosphorylation, yet bound more than 5 times as much DNA as WT STAT5. Therefore, we examined the possibility that these mutations might also be affecting the affinity of DNA binding. However, we found that neither the rates of association or of dissociation of the TD⌬12 mutant are affected when they are compared with those of WT STAT5 (Fig. 6).
How does the C terminus modulate DNA binding activity? We favor the possibility that the C terminus affects the rate of dimer formation. Since only phosphorylated STAT dimers can bind DNA it is possible that certain C-terminal mutations (e.g. TD⌬12), although not affecting the DNA binding affinity per se, result in a shift in the equilibrium toward the phosphorylated dimer, effectively increasing the concentration of STAT5 dimers capable of binding DNA. One potential mechanism might be through the interaction of protein inhibitor of activated STAT (PIAS) proteins (49,50). PIAS proteins associate with the phosphorylated tyrosine of STATs via their SH2 domains and reduce STAT transactivation through inhibition of STAT dimerization and DNA binding. Although a PIAS protein has not been identified for STAT5, it is possible that the mutations that result in enhanced DNA binding activity do so by decreasing the association between PIAS and STAT5. The clon-ing of a STAT5-specific PIAS protein would allow us to directly test this hypothesis.
The S756G mutation within the critical 12-amino acid region resulted in an overall reduction in STAT5 transactivation. Attempts to show phosphorylation at this site in vivo have failed (data not shown), consistent with a previous study on serine phosphorylation of STAT5 proteins (51). Indeed, phosphorylated serine residues other than Ser 756 have been reported for both STAT5a and STAT5b (51). Contrary to what has been reported for STAT-1 and -3 (29,42), mutation of these serine residues had no effect on the transcriptional activity of STAT5a and only a modest effect on STAT5b (51). Since both DNA binding and phosphorylation of Tyr 694 were unaffected by the S756G mutation, the effect of this mutation on transactivation is most likely due to structural changes in the 12-amino acid region.
Although the acidic nature of transactivation domains is important for the activity of many transcription factors (43)(44)(45)(46), we were surprised that the acidic residues within the 12-amino acid region of STAT5 were not essential for transactivation, as mutation of these residues to alanines only reduced transactivation of the GAL4 chimera to about 50% of WT activity in NIH3T3 fibroblasts. Although the mutant GAL4 chimera failed to elicit activity in BaF 3 cells, we believe that this is likely a consequence of reduced protein expression since the acidic mutant was the only mutant for which we have seen FIG. 7. Non-acidic residues, but not the acidic residues within the 12-amino acid region are essential for STAT5 transactivation. A, Amino acid sequence of the 12-amino acid region from WT STAT5. Residues in boldface indicate residues mutated in STAT5 mutants. GAL4-STAT5a cDNAs and pFR-Luc were co-transfected into either BaF 3 (B) or NIH3T3 (C) cells as indicated. Cells were harvested as described in Fig. 2. D, NIH3T3/ProR fibroblasts were co-transfected with either WT or mutant STAT5a cDNAs with (STAT5RE) 6 -61pF* as indicated. 24 h later the culture was divided and continued for 16 -20 h in either the presence or absence of prolactin (1 g/ml). Cells were harvested as described in Fig. 3. B-D, each bar represents the mean Ϯ S.E. of four independent experiments. differential expression. When the identical mutation was introduced in the context of the STAT5 backbone, we found that it led to increased activity in fibroblasts compared with the WT protein. Despite having a reduced Tyr 694 phosphorylation, this mutant exhibited an increased capacity to bind DNA compared with WT STAT5, possibly through augmented dimer formation. Again, a partially active transactivation domain combined with enhanced intrinsic DNA binding result in a net level of activity equivalent to that of WT STAT5.
Our finding that acidic residues are non-essential for transactivation is not without precedent. The same was reported for the glucocorticoid receptor, GAL4, VP16, C1, and v-myb (47,(52)(53)(54)(55). In those cases, hydrophobic residues within the transactivation domain were found to be more critical for transactivation (47,48,54). Consistent with these observations, we found that mutating all the non-acidic residues within the 12-amino acid region resulted in significantly reduced levels of transactivation. When we compared the amino acid sequence of the minimal essential transactivation domain of VP16 with that of 12 amino acids essential for STAT5 transactivation, we found the two sequences to be highly similar, with the conserved sequence EFDLD. Previous studies had shown that both the phenylalanine and leucine in this sequence were essential for transactivation by VP16 (47,48). Mutation of either F751A or L753A in STAT5 severely compromised the ability of the mutant proteins to transactivate, indicating that these residues are crucial for transactivation. Mutating both residues did not result in any further decrease in transcriptional activity, suggesting that Phe 751 may be more critical than Leu 753 .
Transactivation domains are highly divergent regions of transcription factors and tend to be acidic in nature (43,44).
One common feature shared by several acidic transactivation domains, including that of VP16, is that under physiological conditions these regions adopt an unordered structure (55)(56)(57)(58)(59)(60)(61). However, when exposed to more hydrophobic or mildly acidic conditions, they have the propensity to transform into regions rich in secondary structure (55,(57)(58)(59)(60)(61). Only the crystal structure of STATs lacking their C termini has been reported (18,19), suggesting that the transactivation domain of STATs also adopts an unordered structure. Despite this prediction, it is clear from our experiments that a single hydrophobic point mutation in or near the transactivation domain of STAT5 is highly destructive. It is likely that these mutations disrupt secondary structure(s) and affect the ability of this region to contact or stabilize contact with components of either co-activator complexes or of the basal transcriptional machinery. In this regard, these point mutants will help to elucidate the mechanism by which STATs transactivate target genes. Furthermore, these transcriptionally weak mutants will be useful in identifying novel interactors with the C terminus of STAT5 by both biochemical and genetic approaches.  9. Hydrophobic residues Phe 751 and Leu 753 are critical for STAT5 transactivation. A, alignment of the amino acid sequence of the 12-amino acid region from STAT5 with the minimal essential transactivation domain of VP16. Asterisks (*) indicate residues in STAT5 mutated in the TDnon-acid/Ala mutant. Residues underlined in VP16 are essential for transactivation. B, residues in boldface indicate residues mutated in STAT5 mutants. C, GAL4-STAT5 cDNAs and pFR-Luc were co-transfected into BaF 3 cells as indicated. Cells were harvested as described in Fig. 2. Each bar represents the mean Ϯ S.E. of four independent experiments.


extfieldsofstudy: Chemistry Medicine

text:
Rabbit Serum Paraoxonase 3 (PON3) Is a High Density Lipoprotein-associated Lactonase and Protects Low Density Lipoprotein against Oxidation*

The paraoxonase gene family contains at least three members: PON1, PON2, andPON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g.lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.

The paraoxonase gene family in mammals includes at least three members: PON1, PON2 and PON3 (1). The PON genes appear to have arisen by gene duplication of a common evolutionary precursor because they share considerable structural homology and are located adjacently on chromosome 7 in humans and on chromosome 6 in mice (1). PON1, also known as serum paraoxonase/arylesterase (EC 3.1.8.1), is closely associated with high density lipoproteins (HDL) 1 and catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates (2,3). Recently, we reported that purified human and rabbit serum PON1 also hydrolyze of a variety of lactones and cyclic carbonate esters, including naturally occurring lactones and pharmacological agents (4,5). PON1 requires Ca 2ϩ for both its stability and hydrolytic activity (2), and the latter is stimulated by several phospholipids (6). Any physiological role of PON1 is still speculative; however, several lines of evidence suggest that this enzyme protects against atherosclerosis by preventing the oxidation of low density lipoproteins (LDL) (see Refs. 7-9 for reviews). Oxidized phospholipids and/or their degradation/metabolic products may be physiological substrates of PON1 (10). PON1 knockout mice are more susceptible to organophosphate toxicity as well as to atherosclerosis produced by an atherogenic diet (11). PON1 also protects against bacterial endotoxin-induced toxicity (12).
The roles of PON2 and PON3 are much less well understood than are those for PON1. Within a given species, PON1, PON2, and PON3 share about 70% identity at the nucleotide level and about 60% identity at the amino acid level, whereas between the mammalian species particular PONs (1, 2, or 3) share 81-91% identity on the nucleotide level and 79 -90% identity at the amino acid level (1,12). To date, all PON2 and PON3 cDNAs sequenced lack the three nucleotide residues of codon 106 in PON1 (1,12). Some lactone hydrolases previously described in bacteria (13) and in fungi (14) share remarkable structural homology with the PON family members, and we hypothesized that the lactonase activity rather than arylesterase or organophosphatase activities could be a common feature of the PON proteins. In the present study, a rabbit serum lactonase distinct from PON1 was purified and characterized. This is a 40-kDa protein that has an N-terminal sequence (25 amino acids) that exactly matches the deduced amino acid sequence predicted by the rabbit PON3 cDNA we have cloned and that agrees closely with the sequence reported recently for rabbit liver microsomal paraoxonase (MsPON) (15). We defined the substrate specificity of the purified rabbit serum PON3 over a number of esters and lactones and confirmed it with transiently expressed recombinant rabbit PON3. Of considerable interest is the close HDL association of serum PON3 and its enhanced ability compared with rabbit serum PON1 to protect LDL against oxidation.

EXPERIMENTAL PROCEDURES
Rabbit blood was collected by ear vein bleeding and cardiac puncture of anesthetized (ketamine, 40 mg/kg intramuscular, and acetopromazine, 1 mg/kg intramuscular) New Zealand White female rabbits. The blood was allowed to clot for 30 min at room temperature, and the serum was obtained after centrifugation for 15 min at 2,000 ϫ g.
Materials and Chemicals-DEAE Bio-Gel A and SDS-PAGE low range protein molecular weight standards were obtained from Bio-Rad. Sephacryl 200 was purchased from Amersham Pharmacia Biotech. Cibacron Blue 3GA-agarose type 3000, concanavalin A-Sepharose 4B, Tergitol NP-10, and mevastatin (Compactin) were obtained from Sigma. Lovastatin (Mevacor ® ) and simvastatin (Zocor ® ) were purchased as 20-mg tablets from Merck, from which the lactones were extracted with chloroform, evaporated to dryness, and redissolved in methanol. ⑀-Caprolactone was obtained from Acros Chimica (Fair Lawn, NJ). All other lactones and esters used were purchased from Sigma-Aldrich. Acetonitrile high performance liquid chromatography (HPLC) grade was purchased from Mallinckrodt Chemical Works. Glacial acetic acid, methanol, and water (HPLC grade) were obtained from Fisher. Centicons and Centriprep concentrators were purchased from Amicon, Inc. (Beverly, MA).
Enzyme Purification-The initial steps in the purification of rabbit serum PON3 essentially followed the procedure for purification of serum PON1 previously developed in this laboratory (6,16). Rabbit serum was mixed batchwise with Cibacron Blue 3 GA-agarose in 3 M NaCl, 50 mM Tris/HCl buffer (pH 8.0), 1 mM CaCl 2 , and 5 M EDTA, poured into a column, and washed with the same buffer until the A 280 was below 0.3 A units and further with two column volumes of 25 mM Tris/HCl buffer (pH 8.0), 1 mM CaCl 2 . The column was developed with 25 mM Tris/HCl buffer (pH 8.0), 1 mM CaCl 2 , 20% glycerol, and 0.1% sodium deoxycholate; 10-ml fractions were collected at 1 ml/min. Fractions were monitored for both arylesterase activity (phenyl acetate hydrolysis) and lactonase activity (lovastatin hydrolysis) to localize PON1 and PON3, respectively. The two activities co-eluted after Blue-agarose chromatography of serum but were almost completely separated on a DEAE anion exchange column by elution with a linear 300-ml of 0 -175 mM NaCl gradient in TCGT buffer (25 mM Tris/HCl (pH 8.0), 1 mM CaCl 2 , 20% glycerol, and 0.2% non-ionic detergent Tergitol NP-10). Fractions (5 ml) were collected at 0.55 ml/min. Fractions with arylesterase or lactonase activities were purified further separately on a second DEAE column with a NaCl gradient of 0 -120 mM for PON1 and 0 -175 mM for PON3. Fractions with lactonase activity from the second DEAE column were pooled, concentrated 10-fold using a 10-kDa Centricon ultrafiltration unit, and loaded onto a Sephacryl 200 gel filtration column (100 cm ϫ 1 cm). Proteins were eluted with TCGT buffer and 1 ml fractions were collected at 17 ml/h. Fractions with the highest lactonase activities and the lowest contamination with other proteins, as determined by SDSpolyacrylamide gel electrophoresis (PAGE), were pooled and passed through a concanavalin A-Sepharose 4B column. PON3 did not bind to this column and was collected in 25 mM Tris/HCl buffer (pH 7.2), 1 mM CaCl 2 . PON1-containing fractions from a second DEAE column were pooled, loaded on a concanavalin A column, and eluted with a 0 -200 mM ␣-D-mannopyranoside gradient in 25 mM Tris/HCl buffer (pH 7.2), 1 mM CaCl 2 . Finally, the PON1 and PON3 preparations were washed with TCGT buffer using a 50-kDa cut-off Centricon ultrafiltration unit to adjust the buffer and to remove residual concanavalin A fragments. The enzyme preparations were stored at 4°C without substantial loss of activity for more than 3 months.
Protein Assay and SDS-PAGE-Protein was determined by its UV absorption at 280 nm or in samples containing Tergitol NP-10 by the BCA protein assay (Pierce), according to the manufacturer's protocol.
SDS-PAGE was performed as described previously (17), and the gels were stained with Coomassie Blue.
Protein Sequencing-The N-terminal peptide sequencing was performed at the University of Michigan Protein Sequencing Core Laboratory using the Model 494 HT Protein Sequencer (Applied Biosystems Inc., Foster City, CA).
Cloning of Rabbit PON3-Total rabbit liver RNA was isolated using the RNeasy Kit (Qiagen, Valencia, CA) following the supplier's protocol. RT-PCR was performed with the Titan One-tube RT-PCR system (Roche Molecular Biochemicals) according to the manufacturer's instructions. Primers for human PON3 (1), Px3-6 (GGCATAGAACTGT-TCTGGTCCAAGAACC) and Px3-17 (GCTTCTGAAGATATTGATATA-CTCCCCAGTGGGC) were purchased from Midland Certified Reagent Co. (Midland, TX). The RT-PCR products were separated on a 1% agarose gel. Bands of the expected size (as deduced by their similarity to the human PON3) were excised and submitted for sequencing in our DNA Sequencing Core Laboratory. Based on the obtained sequence, primers RPx3-4 (CTCATCTGGTGCAAAGTTTGG) and RPx3-1 (ACA-ACAACGCTCTCTTGTAC) were designed (these and all of the primers below were purchased from Life Technologies, Inc.) and used for amplification of the 5Ј-and 3Ј-ends of rabbit PON3 cDNA using a 5Ј,3Ј-rapid amplification of cDNA ends (RACE) kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Based on the sequence of the new primers of these fragments, RPx3-5 (ATCG-GAA TTCCATGGCGAAGCTCCTGC) and RPx3-6 (AGGCCTCGAGC-TGGAGACTAGAGCAC) were designed and used to amplify the fulllength cDNA. The PCR product (ϳ1200 base pairs) was cloned in the pCR ® II vector with a TOPO ® TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. PON3 clones from at least five animals were sequenced in both directions, and the consensus sequence was submitted to the GenBank TM (accession number AF220944).
Transient Expression of Recombinant Rabbit PON3-The rabbit PON3 clone in pCR ® II vector was digested with EcoRI (Promega) and XhoI (Life Technologies, Inc.) and ligated into a pcDNA3.1(ϩ) expression vector (Invitrogen). The plasmid was propagated in Escherichia coli TOP10 cells (Invitrogen) and then purified using the Qiagen plasmid maxi kit. The insert was sequenced to verify its structure. The highly transfectable 293T/17 human embryonic kidney cell line (ATCC CRL 11268) was used with the permission of David Baltimore (Rockefeller University, New York). Cells were grown in minimum essential medium (Celox Laboratories, Inc., St. Paul, MN) supplemented with 10% heat-deactivated fetal bovine serum (Life Technologies, Inc.) and transfected with 10 g of plasmid DNA as described (18). The medium containing the recombinant enzyme was collected after allowing transfected cells to grow for 4 -5 days. The recombinant PON3 was partly purified using a small Blue-agarose column as described above. The lactonase activity (lovastatin hydrolysis) was used to follow the level of expression/secretion in the culture medium and to localize PON3 in the column fractions.
Hydrolysis of aromatic lactones was monitored by the increase in UV absorbance at 270 nm (dihydrocoumarin), 274 nm (2-coumaranone), and 290 nm (homogentisic acid lactone). In a typical experiment a cuvette contained 1 mM substrate (from a 100 mM stock, dissolved in methanol) in 50 mM Tris/HCl (pH 8.0), 1 mM CaCl 2 , and 5-20 l of enzyme in a total volume of 1 ml. The molar difference extinction coefficients (difference between the absorption coefficients of the acid formed and the lactone) used to calculate the rate of hydrolysis were TABLE I Purification of rabbit serum PON3 Results are given for a typical purification run. Enzyme activity with lovastatin was measured as described under "Experimental Procedures"; yield ϭ (activity of the fractions combined for the next step)/(total activity in serum) ϫ 100 and does not include all of the activity actually recovered. 1295, 876, and 816 M Ϫ1 cm Ϫ1 for dihydrocoumarin, 2-coumaranone, and homogentisic acid lactone, respectively (5). The hydrolysis of other lactones was followed by a colorimetric assay with phenol red (5,19). The reaction cuvette contained 1 mM substrate (from a 100 mM stock solution, dissolved in methanol) in 2 mM HEPES (pH 8.0), 1 mM CaCl 2 , 0.004% (106 M) phenol red, 0.005% bovine albumin, and 5-20 l of enzyme in a total volume of 1 ml. The rate of acid production was followed by monitoring the increase in absorbance at 422 nm. The rate of hydrolysis was derived from a calibration curve obtained using known amounts of HCl as described previously (19).
The enzymatic assays were performed at 25°C using a dual beam Cary 3E UV/visible spectrophotometer (Varian, Australia). Reference cuvettes containing the appropriate buffer plus substrate were used in each assay to correct for any spontaneous hydrolysis.
The hydrolysis of the statin lactones (mevastatin, lovastatin, and simvastatin) was analyzed by HPLC using a Beckman System Gold HPLC with a model 126 programmable solvent module, a model 168 diode array detector set at 238 nm, a model 7125 rheodyne manual injector valve with a 20-l loop, and a Beckman ODS Ultrasphere column (C18, 250 ϫ 4.6 mm, 5 m). In a final volume of 1 ml, 10 -200 l of enzyme and 10 l of substrate solution in methanol (0.5 mg/ml) were incubated at 25°C in 25 mM Tris/HCl (pH 7.6), 1 mM CaCl 2 . Aliquots (100 l) were removed at specified times and added to acetonitrile (100 l), vortexed, and centrifuged for 1 min at maximum speed (Beckman Microfuge). The supernatants were poured into new tubes, capped, and stored on ice until HPLC analysis. Samples were eluted isocratically at a flow rate of 1.0 ml/min with a mobile phase consisting of the following: A ϭ acetic acid/acetonitrile/water (2:249:249, v/v/v) and B ϭ acetonitrile, in A/B ratios of 50/50, 45/55, and 40/60 for mevastatin, lovastatin, and simvastatin, respectively. Under the above conditions the retention times for the carboxylic acid formed and the lactone substrate were as follows: 4.5/6.4 min (mevastatin), 4.4/6.6 min (lovastatin), and 4.8/6.6 min (simvastatin). Response factors for the acid products were calculated from the peak heights after complete alkaline hydrolysis of the lactones in 0.02 M NaOH.
Separation of Rabbit Serum Lipoproteins by Gel Filtration-Serum was loaded onto a Bio-Gel A-15m column (36 ϫ 1.8 cm) and eluted with 50 mM Tris HCl (pH 8.0), 1 mM CaCl 2 . Fractions were collected and analyzed on a Hitachi 912 autoanalyzer (Roche Molecular Biochemicals) for phospholipids, triglycerides, and total and esterified cholesterol using commercially available kits from Wako Bioproducts (Richmond, VA). Representative fractions were combined, concentrated, and analyzed by high performance gel filtration chromatography (HPGC) to determine the lipoprotein cholesterol distribution as described elsewhere (20).
Lipoprotein Separation by Ultracentrifugation of Serum-Fractions were prepared by sequential flotation ultracentrifugation to isolate very low density lipoproteins (VLDL) (d Ͻ1.019 g/ml), LDL (d ϭ 1.019 to 1.063 g/ml), and HDL (d ϭ 1.063 to 1.2 g/ml) (21). Lipoprotein fractions were dialyzed against phosphate-buffered saline (pH 7.4) containing 1 mM CaCl 2 at 4°C for 24 h, then kept at 4°C in the dark. The isolated fractions were analyzed within the next 3-4 days for enzymatic activities and by HPGC to confirm their identity and purity as described (20).
Copper-induced LDL Oxidation in Vitro-Human LDL was isolated by ultracentrifugation as described above and used within the next 3-4 days. LDL (0.1 mg protein/ml) oxidation was induced with 2-10 M CuSO 4 in phosphate-buffered saline (pH 7.4) in the absence or presence of PON3 or PON1 for up to 5 h at 37°C. The kinetics of lipoprotein oxidation was followed by monitoring conjugated diene formation at 234 nm (22) using a quartz microtiter plate in a SPECTRAmax ® 190 plate reader (Molecular Devices, Sunnyvale, CA). The lag time was estimated by drawing a perpendicular line to the x axis from the intersection of straight lines drawn through the absorption curves during the lag phase and the propagation phase, as illustrated on Fig. 4A. Lipoprotein oxidation was also determined by the lipid peroxides test, which analyzes lipid peroxides by their capacity to convert iodide to iodine, as measured photometrically at 365 nm (23).

Purification of a 40-kDa Protein with Lactonase Activity-
The purification of PON3 from rabbit serum is summarized in Table I. We used lovastatin hydrolysis (lactonase activity) and phenyl acetate hydrolysis (arylesterase activity) to follow purification of PON3 and PON1, respectively. During the Blueagarose chromatography of serum, both activities co-eluted (Fig. 1A) but were resolved by DEAE anion exchange chroma-tography (Fig. 1B). Fractions with peak arylesterase activity were eluted at 87 mM NaCl and fractions with peak lactonase activity were eluted at 105 mM NaCl, respectively. The lactonase activity was further purified through a second DEAE and a Sephacryl 200 gel filtration column and correlated with the intensity of a 40-kDa protein on SDS-PAGE. Two other pro- teins with molecular masses of 50 and 63 kDa co-purified through all steps and became enriched during the purification (Fig. 1C, 2nd lane). The three proteins were transferred to polyvinylidene difluoride membrane, and each was submitted for N-terminal sequencing. The resolved peptide sequences are presented on Table II. The N terminus of the 40-kDa protein is identical with the deduced amino acids sequence of the rabbit PON3 cDNA we cloned (see below) and 96% identical with the amino acid sequence of MsPON (15). The N terminus of the 50-kDa protein is 73% identical with the deduced sequence (residues 22 to 36) of the human platelet-activated factor acetyl hydrolase (PAF-AH) precursor (25). The N-terminal amino acid of the purified human PAF-AH is Ile-42, and the estimated size of the enzyme on SDS-PAGE is 44 -45 kDa (25). Thus, the rabbit analogue of PAF-AH has 20 additional N-terminal amino acids, which could explain the observed difference in the molecular mass. The N terminus of the 63-kDa protein is 93% identical with both human and mouse vanin 1 (26). Both PAF-AH and vanin proteins are N-glycosylated (25,26) and were retained by the concanavalin A column we used as a final step in the purification of PON3. The lactonase activity did not bind to the column, and the washout fractions showed a single band on SDS-PAGE with trace contamination of albumin and concanavalin A fragments (Fig. 1C, lanes 3-5).
The isolation of PON3 from its natural environment and the use of detergents, which was unavoidable because of the tight association of PON3 with other proteins, led to a decrease in its FIG. 2. Nucleotide and amino acid sequences of rabbit PON3. The deduce amino acids sequence is numbered from the initial methionine. The asterisks for the amino acid at position 106 as well as for the nucleotides in the corresponding codon are included in accordance to the recommended nomenclature system for the paraoxonases (27). The N-terminal polypeptide sequenced directly is italicized. The differences with MsPON (12) are shown below the corresponding amino acids.  specific activity. Phospholipids, which were shown to stimulate the hydrolytic activity of purified PON1 (6), e.g. dilaurylphosphatidylcholine, were without effect on the PON3 lactonase activity (lovastatin hydrolysis), but adding apolipoprotein A-1 (apoA-1) in eighth-fold excess doubled it (data not shown).
Cloning of the Rabbit PON3 cDNA-We used the human PON3 sequence as a model for retrieving the rabbit PON3 cDNA as outlined under "Experimental Procedures." We cloned a 1194-base pair cDNA with a 1065-base pair open reading frame (Fig. 2), encoding a protein of 39.5 kDa. This predicted mass is consistent with that determined for the purified lactonase by SDS-PAGE. In the mature protein, as previously shown for the rabbit and human PON1s (24), only the initial methionine is processed, and the remainder of the leader sequence is retained. The rabbit PON3 nucleotide sequence is 86 and 79% identical with those of human and mouse PON3, respectively, as well as 82 and 78% similar to their deduced amino acid sequences. Rabbit PON3 is 64/58% identical with the rabbit PON1 nucleotide/amino acids sequences (24). We cloned and sequenced rPON2 2 and found it 68/65% identical with rPON3 nucleotide/amino acids sequences.
Characterization of the Purified Rabbit Serum PON3-The purified PON3 hydrolyzed lovastatin with a specific activity of about 130 nmol/min/mg of protein. PON3 required Ca 2ϩ for this hydrolytic activity, which could be completely inhibited by EDTA. The lactonase activity was practically unaffected by millimolar concentrations of diisopropyl fluorophosphate or paraoxon (data not shown). The pH optimum for the lovastatin hydrolysis was estimated to be between pH 7.0 and 7.6 (data not shown). The rates of hydrolysis of simvastatin (73 nmol/ min/mg) and mevastatin (95 nmol/min/mg of protein) were within the same range as lovastatin. In contrast, the hydrolytic activity of purified rabbit serum PON1 with lovastatin was less than 0.2 nmol/min/mg.
A comparison of purified rabbit serum PON3 and PON1 hydrolytic activities is presented in Table III. Phenyl acetate and thiophenyl acetate were poor substrates for PON3, whereas the rate of hydrolysis with dihydrocoumarin, 2-coumaronone, and some 5-and 6-member ring lactones with aliphatic substituents was up to 2 orders of magnitude higher. In contrast to PON1, PON3 had no activity with unsubstituted lactones or with 4-and 7-member ring lactones. PON3 required at least one methylene group in the 5-or 6-position (for the ␥or ␦-lactones, respectively), and its lactonase activity increased with the number of carbon atoms in the aliphatic chain (up to 5 for the ␥-lactones and up to 7 for the ␦-lactones). Compared with PON3, rabbit PON1 exhibited more potent lactonase activity and broader substrate specificity, but no simple trait in the substrate structure-enzyme activity relationship could be identified. Thus, the PON1/PON3 lactonase activity ratio varied from 0.9 -4-fold up to 52-97-fold (Table III). Paraoxon was not hydrolyzed by PON3.
Transient Expression of Recombinant Rabbit Pon3-293T/17 cells transfected with rabbit PON3 cDNA in pcDNA3.1(ϩ) expressed and secreted PON3, as evaluated by the increase in the lactonase activity (lovastatin hydrolysis) of the expression medium. The highest lactonase activity (2.8 Ϯ 0.2 nmol/ml of medium, average from four 10-cm culture dishes) was obtained 4 -5 days after transfection. The expression medium from 293T/17 cells transfected only with the pcDNA3.1(ϩ) vector had no lactonase activity. The activity of the lysate from PON3transfected cells harvested 5 days after transfection accounted for only 25% of the total lactonase activity, suggesting efficient secretion of the enzyme. Expression medium from PON3-trans-fected cells was chromatographed on a small Blue-agarose column. The recombinant PON3 co-eluted with apoA-1 and albumin (from the supplemented medium) and had the same apparent molecular mass on SDS-PAGE as PON3 purified from rabbit serum (data not shown). The hydrolytic activity of the Blue-agarose fraction, containing the recombinant PON3, was 6.5 nmol/min/ml (lovastatin), 4.6 mol/min/ml (dihydrocoumarin), and 0.043 mol/min/ml (phenyl acetate). Paraoxon was not hydrolyzed by this fraction.
HDL Association of PON3-The co-purification of PON3 with apoAI and PON1 through the Blue-agarose chromatography step (Fig. 1A) suggested that both PONs are associated with the serum HDL. In another experiment, rabbit serum lipoproteins were separated by gel filtration chromatography. VLDL eluted in fractions 10 -14, LDL in 18 -28, and HDL in 29 -52, respectively (Fig. 3A). The purity and the identity of the lipoprotein fractions were evaluated by HPGC of the lipoprotein cholesterol (Fig. 3B). The arylesterase activity was recovered in fractions 30 -46, and the lactonase activity (lovastatin) was recovered in fractions 29 -49 (Fig. 3C). Thus, both activities co-eluted with the HDL fractions.
Rabbit serum lipoproteins were also separated by sequential flotation ultracentrifugation (Table IV.). Virtually all of the recovered lactonase activity (PON3) as well as all arylesterase and paraoxonase activities (PON1) were in the HDL fraction. The higher recovery of PON3 (22%) compared with PON1 (13%) during ultracentrifugation could be due to tighter binding to HDL and/or greater stability of PON3.
Protection of LDL against Lipid Peroxidation-The protective role of purified human PON1 against in vitro copperinduced oxidation of HDL and LDL has been well established (7-10, 12, 28). Initial experiments in our laboratory showed 2 C. Watson, unpublished data.

DISCUSSION
Purified rabbit serum PON3 has a molecular mass of 40 kDa on SDS-PAGE, in agreement with the predicted value from the deduced amino acid sequence of the PON3 cDNA. Like PON1, rabbit serum PON3 retains most of its hydrophobic leader sequence, and only the initial methionine is cleaved. This portion of PON1 is responsible for its tight binding to HDL phospholipids (29), and it is reasonable to assume that this also accounts for the association of rabbit serum PON3 with HDL. In this study we separated rabbit serum lipoproteins by gel chromatography and by ultracentrifugation. Virtually all of the PON3 hydrolytic activity was recovered with the HDL fractions ( Fig. 3 and Table IV). Immunopurified PON1 is associated with two major proteins: apoA-I and apoJ (30,31), but PON3 seems to be more widely distributed than PON1 in the gel filtration HDL fractions (Fig. 3). Further experiments will be needed to study the association of PON3 with particular HDL subclass and/or particular apolipoproteins. Rabbit liver MsPON appears to lack the tripeptide Ala-Lys-Leu at its N terminus (15). At present, it is not clear whether this difference is an artifact arising during purification or the result of different processing. Thirteen additional differences were found between the reported MsPON amino acid sequence (15) and that deduced from rabbit PON3 cDNA (Fig. 3). Recently, we observed that commercially available New Zealand White rabbits show at least eight-nucleotide differences in the coding region of the PON1 gene, resulting in five amino acid changes as well as in a polypeptide four amino acids shorter at the C terminus. 3 A similar number of changes in the neighboring PON3 gene and/or an additional PON3 gene in rabbits may account for the differences between the PON3 sequence we found and that reported for MsPON (15).
Rabbit serum PON1 and PON3 share considerable amino acid sequence identity (58%) but show distinct substrate specificities. PON1 hydrolyzes paraoxon and aromatic esters (2,4) as well as many aromatic and aliphatic lactones (Refs. 4 and 5 and this study). Although the active site of the PON1 has not been established, some residues essential for the hydrolytic 3 C. Watson, personal communication. activity have been identified by both chemical modification and site-directed mutagenesis (32)(33)(34). The chemical modification of tryptophan, histidine, aspartic acid/glutamic acid, or cysteine residues reduced or abolished enzymatic activity. Sixteen of these residues in human PON1 were identified as essential for the hydrolytic activity, and all of them except His-243 and Trp-281 are conserved in rabbit PON3. The two PON1 mutants, W281L and H243K, corresponding to the PON3 sequence, have markedly reduced arylesterase and organophosphatase activity (32,33). This observation may at least in part explain the limited arylesterase and paraoxonase activity of rabbit serum PON3 we observed. Both PON3 and PON1 have lactonase activity, but PON1 hydrolyzes a much broader spectrum of lactones at higher rates than PON3. Six-member ring lactones were better substrates than their five-member homologs for both PON3 and PON1. Interestingly, rabbit serum PON3 hydrolyzes the statin lactones at a much higher rate than PON1. During the purification of rabbit serum PON3 and PON1, we found that the PON1 is about 200 times more abundant than PON3, but nevertheless, the overall contribution of PON1 to rabbit serum lactonase activity (lovastatin hydrolysis) is less than 1%, as calculated from the specific activity of the purified enzyme. It has been shown that lovastatin and simvastatin are rapidly hydrolyzed by mouse and rat plasma and very slowly by human and dog plasma (35,36), whereas simvastatin is hydrolyzed by rabbit plasma at rates between those of rodent and the human plasma (36). However, we found that about 95% of the mouse and rat serum lactonase activity (with lovastatin) does not bind to Blue-agarose and is completely inhibited by diisopropylphosphofluoridate or paraoxon but not by EDTA. 4 Thus, it is more likely that in rodents carboxylesterase(s) account for this activity. It has been shown that at least three distinct esterases in humans are capable of lovastatin hydrolysis, one in the plasma and two in the liver (37). Purified human serum PON1 hydrolyzes the statin lactones at rates close to the purified rabbit serum PON1 (13), and statin hydrolysis by human serum is only about twice that calculated from the activity of purified human PON1. Thus, if PON3 is present in rodent and human serum, it is even less abundant than in rabbit serum and/or has lower lactonase activity with the statin prodrugs than rabbit serum PON3.
A physiological role for PON1 and the other PON enzymes is still unknown. However, epidemiological and biochemical evidence is accumulating that suggests an important role for PON1 against atherosclerosis (7)(8)(9). PON1 protects both LDL and HDL from oxidation (28,38,39), probably related to its ability to hydrolyze specific oxidized phospholipids (7, 10) and cholesteryl linoleate hydroperoxides (28,39,40). The ability of PON1 to protect LDL against oxidation is paralleled by inactivation of the enzyme, which results from an interaction of oxidized phospholipids and cholesteryl esters with the PONfree sulfhydryl group (39). Although human and rabbit PON1 have about the same ability to protect LDL against in vitro copper-induced oxidation, 4 we unexpectedly found that rPON3 is about 100-fold more potent than rPON1 (Fig. 4). We propose FIG. 4. Effect of purified PON3 and PON1 on in vitro copperinduced LDL oxidation. LDL (100 g/ml) were incubated at 37°C in phosphate-buffered saline (pH 7.4) in the presence or absence of purified PON3 or PON1 as indicated in the legends. Lipid peroxidation was initiated with 5 M CuSO 4 . A, accumulation of conjugated dienes measured as an increase in the absorbance of the samples at 234 nm after the addition of Cu 2ϩ and calculated lag times (inset panel). B, effect on lipid peroxide formation. Lipid peroxides were measured as described in El-Saadani et al. (23). The data are shown as the mean Ϯ S.D. for triplicates from a representative out of three experiments with similar results. There were no detectable levels of lipid peroxides in the LDL before oxidation. * and ** denote significant difference from the control at p Ͻ 0.05 and p Ͻ 0.01, respectively.

TABLE IV
Enzyme activities of rabbit serum lipoprotein fractions, separated by ultracentrifugation Lipoprotein fraction preparation and enzyme activity assays were performed as described under "Experimental Procedures." HPGC was performed according to Kieft et al. (20); the distribution of the lipoprotein cholesterol in serum is shown with a contour on each panel overlaid with the cholesterol peak in each fraction (shaded area). that rPON3 acts by a mechanism similar to that of PON1. Further experiments are needed to find the reason(s) for better protection by rPON3 against LDL oxidation, which might be due to different substrate specificity, kinetic properties and/or stability of the two enzymes. We estimate that rPON3 is present in serum at concentrations of about 20 -50 nmol/liter, whereas the concentration of rPON1 in serum is about 5 mol/ liter. If the in vitro data we report here reflects the in vivo activity of PON3 in rabbits, its contribution against LDL oxidation might be as great as that of rPON1.
Northern blot analyses had previously detected PON1 mRNA only in rabbit and human liver (24). Using RT-PCR, PON1 mRNA was detected in adult mouse liver, kidney, heart, brain, small intestine, and lung but not in pancreas nor in white blood cells (1), whereas PON2 and PON3 mRNAs were detected in all of the tissues studied (1). 4 Based on the expressed sequence tag clones representation in GenBank TM , it appears that PONs differ in their tissue expression and distribution; human and mice PON1-expressed sequence tags originate from liver, lung, and brain, PON2-expressed sequence tags are expressed ubiquitously, and PON3 is represented somewhere in between. To date, there are no data about the secretion of PON2 and PON3 in other species than rabbit (this report).