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Genes to Cells (2007) 12, 1153-1161. doi:10.1111/j.1365-2443.2007.01126.x
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Type I platelet-activating factor acetylhydrolase catalytic subunits over-expression induces pleiomorphic nuclei and centrosome amplification

 Noritaka Yamaguchi1,2, Hiroyuki Koizumi1, Junken Aoki1,3, Yumiko Natori2, Kiyotaka Nishikawa2, Yasuhiro Natori2, Yasukazu Takanezawa1 and Hiroyuki Arai1,*

1 Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
3 PRESTO, Japan Science and Technology Corporation, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
LIS1, a causative gene product for type I lissencephaly, binds to and regulates the dynein motor and the centrosome. LIS1 also forms a complex with the catalytic subunits {alpha}1 and {alpha}2 of type I platelet-activating factor acetylhydrolase [PAF-AH (I)]. However, the cellular function of the catalytic subunits remains unknown. In this study, we showed that over-expression of the catalytic subunits, especially {alpha}2, in cultured cells induced dramatic phenotypical changes including nuclear shape change, centrosomal amplification and microtubule disorganization. We examined if these effects were due to the catalytic activity and/or binding of {alpha}2 to LIS1. Substitution of a single amino acid Glu39 of murine {alpha}1 and {alpha}2 by Asp ({alpha}2-E39D) did not affect catalytic activity but completely abolished LIS1 binding. Over-expression of either {alpha}2-E39D or the catalytically inactive {alpha}2-S48C revealed that {alpha}2-E39D, but not {alpha}2-S48C, lost its ability to induce above-mentioned phenotypic changes. Biochemical analyses showed that LIS1 present in the precipitate fraction of murine brain homogenates could be translocated to the soluble fraction by {alpha}2, but not by {alpha}2-E39D. These results suggest that over-expression of the PAF-AH (I) catalytic subunits induces centrosomal amplification and microtubule disorganization by disturbing intracellular localization of LIS1.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
LIS1 is the causative gene for lissencephaly type I (Reiner et al. 1993; Hattori et al. 1994a), a brain malformation characterized by a smooth surface of the cortex. This disorder is due to a defect in neuronal migration during brain development (Dobyns et al. 1993). Mice homozygous for the LIS1 null mutation die early, soon after implantation (Hirotsune et al. 1998; Cahana et al. 2001). Heterozygous and compound heterozygous mice have expression level-dependent defects in neuronal migration (Hirotsune et al. 1998). LIS1 interacts with a number of proteins, including tubulin (Sapir et al. 1997), cytoplasmic dynein (Faulkner et al. 2000; Smith et al. 2000), NUDE (Feng et al. 2000; Kitagawa et al. 2000), NUDEL (Niethammer et al. 2000; Sasaki et al. 2000) and NUDC (Aumais et al. 2001). Through interaction with these proteins, LIS1 plays an important role in microtubule-associated cellular functions such as mitotic cell division, chromosomal segregation and neuronal migration.

LIS1 is also a non-catalytic subunit of type I platelet-activating factor acetylhydrolase [PAF-AH (I)] (Hattori et al. 1994a). PAF-AH (I) contains a dimer of two catalytic subunits, {alpha}1 and {alpha}2, to which LIS1 binds (Hattori et al. 1993, 1994a,b, 1995). The {alpha}1 and {alpha}2 catalytic subunits belong to a novel serine esterase family, show ~60% amino acid homology with each other, and form homodimers or a heterodimer depending on the tissue and embryonic stage of development (Manya et al. 1998). The tertiary structure of the {alpha}1/{alpha}1 homodimer shows close resemblance to G{alpha} subunits of heterotrimeric G proteins (Ho et al. 1997). Crystal structure analysis of the PAF-AH (I) complex has revealed that the LIS1 homodimer binds symmetrically to one {alpha}2/{alpha}2 homodimer via the highly conserved top faces of the LIS1 ß propellers (Tarricone et al. 2004).

Homologues of LIS1 have been identified in a wide range of organisms, including Saccharomyces cerevisiae (Geiser et al. 1997), Aspergillus nidulans (Xiang et al. 1995), Caenorhabditis elegans (Dawe et al. 2001), Drosophila melanogaster (Sheffield et al. 2000) and mammals. On the other hand, homologues of the catalytic subunits have been identified only in higher eukaryotes (Sheffield et al. 2000). PAF-AH (I) catalytic subunits as well as LIS1 are expressed at high levels in both brain and testis (Koizumi et al. 2003). The expression levels of the PAF-AH (I) catalytic subunits and LIS1 in murine tissues are proportional to the expression of {alpha}-tubulin, a component of microtubules (Koizumi et al. 2003), suggesting that the catalytic subunit is also involved in microtubule dynamics together with LIS1. Unexpectedly, in a catalytic subunit knockout mouse model ({alpha}1–/–/{alpha}2–/–), the brain showed no obvious abnormalities in neuronal lamination, but the testis, expressing catalytic subunits and LIS1 abundantly, showed severe spermatogenesis impairment (Koizumi et al. 2003; Yan et al. 2003). Moreover, LIS1 protein levels, but not mRNA levels, are significantly reduced in {alpha}2–/– and {alpha}1–/–/{alpha}2–/– mice (Koizumi et al. 2003). These observations led us to speculate that the catalytic subunits play an important role in microtubule-associated cellular functions together with LIS1. In several human lymphomas, the {alpha}2 gene is disrupted by chromosomal translocation of the first intron (Lecointe et al. 1999). This recombination event does not affect the coding sequences of the gene but removes the first non-coding exon and places the remaining exons under the control of the immunoglobulin heavy chain regulatory element (Meerabux et al. 1994), suggesting that the chromosomal translocation leads to over-expression of {alpha}2 in human lymphomas.

In this study, we performed an over-expression study using Chinese hamster ovary (CHO) cells in culture and found that over-expression of the catalytic subunits, especially {alpha}2, induced centrosomal amplification and microtubule disorganization, phenomena frequently occurring in human cancer (Kawamura et al. 2004).


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Over-expression of the catalytic subunits in living cells induces pleiomorphic nuclei and centrosomal amplification

In order to gain more insight into the cellular function of the PAF-AH (I) catalytic subunits, over-expression studies were performed using CHO cells. We initially found that cytomegalovirus (CMV) promoter-driven transient over-expression of the catalytic subunits induced significant cellular toxicity (data not shown). Then, transfectant CHO cells were generated expressing murine {alpha}1 or {alpha}2 subunits under the control of the tetracycline-responsive promoter (Tet-off). These established cell lines showed no obvious phenotypic changes as far as tetracycline was present in the culture medium. Upon removal of tetracycline from the medium, these CHO clones accumulated exogenous {alpha}1 or {alpha}2 subunits to approximately fivefold levels over background (Fig. 1A). Interestingly, analysis of the cellular microstructure revealed severe impairment such as cell enlargement, structural changes of the nucleus and microtubule disorganization, primarily when the {alpha}2 subunit was over-expressed (Fig. 1B–D, B'–D'). Staining of the centrosome or the microtubule organizing center (MTOC) with {gamma}-tubulin revealed that most cells with normal expression of the PAF-AH (I) catalytic subunit had a single centrosome (Fig. 1B'), while all cells with over-expression of {alpha}2 had more than two centrosomes (Fig. 1D'). Phenotypical changes were far less in cells over-expressing {alpha}1 (Fig. 1C,C') than in cells over-expressing {alpha}2 (Fig. 1D,D'). Nonetheless, when compared with control cells, {alpha}1 over-expression led to visible enlargement of the nucleus as well as to an increase of centrosomes to two or more per cell. Thus, over-expression of PAF-AH (I) catalytic subunits led to the generation of pleiomorphic nuclei and centrosome amplification.


Figure 1
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  		Figure 1  Over-expression of {alpha}2 causes pleiomorphic nuclei and centrosome amplification in CHO cells. (A) CHO Tet-off cells were cultured in the presence or absence of 2 µg/mL tetracycline for 48 h before being lysed and checked for the expression of {alpha}1 and {alpha}2 by Western blotting using anti-{alpha}1 or anti-{alpha}2 antibody. (B–D, B'–D') CHO Tet-off cells were cultured in the presence (B, B') or absence (C, C', D, D') of 2 µg/mL tetracycline for 48 h before being fixed with paraformaldehyde or methanol. (B–D) Paraformaldehyde-fixed cells were stained with anti-{alpha}-tubulin antibody (green) to reveal the microtubule structure and counterstained with DAPI (cyan). (B'–D') Methanol-fixed cells were stained with anti-{gamma}-tubulin antibody serving as a centrosomal marker.

 
In contrast to normal CHO cells (Fig. 2A–D), severe mitotic spindle defects were seen during mitosis in many cells over-expressing {alpha}2, including multipolar or mispositioned spindles (Fig. 2E–H, arrows). Supernumerary poles were uneven in size, often with two major poles that defined a major spindle axis, and a small extrapole, suggesting uneven fragmentation of one pole. In these cells, chromosome alignment was perturbed in the metaphase (Fig. 2F), but these chromosomes still moved towards these abnormally formed multiple spindle poles (Fig. 2G,H). Presumably, these abnormal chromosome alignments resulted in a generation of pleiomorphic nuclei in cells over-expressing {alpha}2.


Figure 2
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  		Figure 2  Over-expression of {alpha}2 induces spindle abnormalities. CHO Tet-off cells in the presence (A–D) or absence (E–H) of 2 µg/mL tetracycline for 48 h were immunostained with anti-{alpha}-tubulin antibody (green) to reveal the microtubule structure and counterstained with propidium iodide (red). (A, E) Late prometaphase, (B, F) methaphase, (C, G) anaphase, (D, H) early telophase. Arrows indicate abnormal mitotic spindle poles in {alpha}2-over-expressed CHO cells.

 
Identification of the critical amino acid for LIS1-binding in the PAF-AH (I) catalytic subunits

To examine if the phenotypes of CHO cells over-expressing PAF-AH (I) catalytic subunits result from the catalytic activity and/or binding to endogenous LIS1, we constructed a mutant {alpha}2 unable to perform either function. Since we had already identified the catalytic center of {alpha}2 as Ser48, we constructed a catalytically inactive {alpha}2-S48C in which Ser48 was substituted by Cys and found that this mutant {alpha}2 could interact with LIS1 (data not shown).

Then we tried to construct the mutant {alpha}2 that is unable to interact with LIS1 without affecting catalytic activity. Tarricone et al. (2004) have recently determined the crystal structure of the LIS1/({alpha}2/{alpha}2) complex and identified eleven residues involved in LIS1 binding in each {alpha}2 subunit. We undertook a different approach to identify the critical amino acid(s) for LIS binding. To identify the amino acids responsible for LIS1 binding, we selected four regions according to the following criteria: (i), the region should be on the surface of the {alpha}2/{alpha}2 homodimer (Ho et al. 1997); (ii), the amino acid sequence should not be similar to that of the Drosophila homologue, since the Drosophila homologue is known to not bind to LIS1 (Sheffield et al. 2000); and (iii) the amino acid sequences of {alpha}1 and {alpha}2 should be very similar, since both {alpha}1 and {alpha}2 bind LIS1. The four regions (15–40, 121–133, 145–160 and 161–185) of murine {alpha}2 were replaced with the corresponding sequences of the Drosophila homologue, and the resultant myc-tagged chimeric proteins were expressed together with LIS1 in CHO cells to elucidate their interaction. Among the chimeric proteins tested, only the chimeric protein that contained Drosophila amino acids 10–35 instead of mammalian amino acids 15–40 showed no affinity to LIS1 (Fig. 3A, left panel). We divided the relevant region into two amino acid sequences, namely: sequence 15–28 and 29–40 and found that the {alpha}2 chimeric protein (15–28) was still capable of LIS1 binding, while alteration of the amino acid sequence 29–40 to the Drosophila sequence resulted in complete loss of binding (Fig. 3A, left panel). This chimera showed full PAF-AH activity (Fig. 3A, right panel). We then focused on the differences between mammals and Drosophila regarding each amino acid in the region 29–40. One or two of the amino acids each were replaced by the corresponding amino acids of the Drosophila sequence and the ability of the mutant {alpha}2 to bind LIS1 was checked. Amazingly, the single substitution of Glu39 by Asp (to form {alpha}2-E39D) led to a complete loss of LIS1 binding, while the substitution of all other amino acids in the defined region had no influence on LIS1 binding capacity (Fig. 3B, left panel). {alpha}2-E39D had full PAF-AH activity (Fig. 3B, right panel). Furthermore, when recombinant {alpha}2-E39D obtained from E. coli was applied to a gel filtration column, a dimer with a molecular weight of 50–60 kDa was eluted (data not shown). Thus, the single substitution of Glu39 by Asp did not appear to induce a significant conformational change of the three-dimensional structure, although LIS1 binding was drastically reduced.


Figure 3
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  		Figure 3  CHO cells were transiently co-transfected with pcDNA3/LIS1 and with each of the myc-tagged murine {alpha}2 catalytic subunits and chimera mutants. The chimeras of 15–40, 15–28 and 29–40 are the construct in which each murine {alpha}2 sequence was substituted by 10–35, 10–23 and 24–35 of the Drosophila {alpha} homologue, respectively. V33I, L34S is a construct in which both Val33 and Lue34 were substituted by Ile and Ser, respectively. K36R, D37E is a construct in which both Lys36 and Asp37 were substituted by Arg and Glu, respectively. Whole-cell extracts were made 48 h after transfection in cell lysis buffer (see Experimental procedures). Immunoprecipitaion was performed with a monoclonal anti-myc antibody. The immunoprecipitates were dissolved in SDS sample buffer and analyzed by Western blotting by anti-myc or anti-LIS1 antibodies. PAF-AH activity of each of the myc-tagged catalytic subunits were quantitated as described in Experimental procedures. The results are expressed as % control ± SD (n = 3).

 
Binding of the catalytic subunit to LIS1 leads to centrosomal and microtubular abnormalities

We then over-expressed {alpha}2-S48C with no catalytic activity and {alpha}2-E39D with no LIS1 binding activity in CHO cells (Fig. 4A). The phenotypical changes described above were still observed when {alpha}2-S48C was over-expressed (Fig. 4B,B'), indicating that the catalytic activity of the protein was not linked to the observed structural changes in the cell. In contrast, when {alpha}2-E39D was over-expressed in CHO cells instead of wild-type {alpha}2, no phenotypical changes were observed in the mutant {alpha}2-E39D (Fig. 4C,C'), showing that binding of the {alpha}2 catalytic subunit to LIS1 is a prerequisite for structural cell abnormality.


Figure 4
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  		Figure 4  Over-expression of {alpha}2-E39D does not cause pleiomorphic nuclei and centrosome amplification in CHO cells. (A) CHO Tet-off cells were cultured in the presence or absence of 2 µg/mL tetracycline for 48 h before being lysed and checked for the expression of {alpha}2 by Western blotting with anti-{alpha}2 antibody. (B, B', C, C') CHO Tet-off cells were cultured without tetracycline for 48 h before being fixed with paraformaldehyde or methanol. (B, C) Paraformaldehyde-fixed cells were stained with anti-{alpha}-tubulin antibody (green) to reveal the microtubule structure and counterstained with DAPI (cyan). (B', C') Methanol-fixed cells were stained with anti-{gamma}–tubulin antibody serving as a centrosomal marker.

 
Translocation of LIS1 in the precipitates to the soluble fraction by the catalytic subunits

Interaction of LIS1 with the PAF-AH (I) catalytic subunits was also examined in vitro. Murine brains were homogenized and then centrifuged at 100 000 g to separate soluble and precipitate fractions. As shown in Fig. 5A, LIS1 was detected in both the soluble and precipitate fractions, while PAF-AH (I) catalytic subunits were detected solely in the soluble fraction. Due to the fact that most {gamma}-tubulin (a component of the centrosome) and p150Glued (a component of the dynein motor complex) were found in the 100 000 g precipitate fraction (Fig. 5A), we assumed that LIS1 present in the 100 000 g precipitate fraction was the one interacting with the centrosome and/or the dynein motor complex.


Figure 5
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  		Figure 5  Recombinant {alpha}2 translocates LIS1 from the murine brain precipitate fraction to the soluble fraction. (A) Western blotting analysis of {alpha}2, LIS1, NUDEL, {gamma}-tubulin and p150Glued in adult murine brain cytosol fraction (sup) and precipitate (ppt) fraction. (B) Insoluble (precipitate) components prepared from adult murine brain were re-homogenized with SET buffer, and purified GST-{alpha}2 (wild-type or E39D mutant) recombinant protein was added. After a 1-h incubation, the mixture was centrifuged at 100 000 g and the supernatants and precipitates were assayed for Western blotting analysis with anti-LIS1 or anti-NUDEL antibodies.

 
The 100 000 g precipitate fraction was resuspended in a buffer and incubated with or without recombinant {alpha}2 or {alpha}2-E39D at 4 °C for 1 h. The mixtures were then centrifuged again at 100 000 g to separate the soluble and precipitate fractions. Even in the absence of {alpha}2, a non-negligible amount of LIS1 was detected in the soluble fraction, while addition of recombinant {alpha}2 resulted in a major shift of LIS1 to the soluble fraction (Fig. 5B). On the other hand, addition of recombinant {alpha}2-E39D did not redistribute LIS1 to the soluble fraction. We therefore conclude that LIS1, which is localized in the precipitate fraction most likely by interacting with the centrosomes and the dynein motor complex, can be extracted into the soluble fraction via {alpha}2.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we found that over-expression of PAF-AH (I) catalytic subunit, especially {alpha}2 in CHO cells resulted in cell abnormalities, such as pleiomorphic nuclei and centrosome amplification. However, over-expression of {alpha}2-E39D, which is unable to interact with LIS1, caused no abnormality in cell structure or LIS1 localization. In a separate in vitro experiment, we showed that LIS1 in the precipitate fraction of murine brain homogenates possibly in association with the centrosome and/or the dynein motor complex could be extracted into the soluble fraction by addition of {alpha}2 recombinant proteins, but not by {alpha}2-E39D. These data suggest that over-expression of {alpha}2 in cultured cells caused redistribution of LIS1 from the centrosome and/or the dynein motor complex to the cytosol where abundant {alpha}2 is present and that this LIS1 mis-localization induced the observed phenotypical abnormalities. Centrosome amplification is known to frequently occur in human cancer (Kawamura et al. 2004). As mentioned earlier, the {alpha}2 expression is thought to be up-regulated by chromosomal translocation in certain human lymphomas (Lecointe et al. 1999). Our finding that over-expression of {alpha}2 in the cell induces centrosome amplification suggests that this phenomenon is a major cause of cellular transformation occurring in the lymphoma cases mentioned above.

It is well established that perturbation of LIS1 levels also causes centrosome malfunction and impaired cell division. Defects in Pac1, the yeast LIS1 homologue, cause mitotic spindle abnormalities (Geiser et al. 1997). LIS1 homozygous-null C. elegans mutants exhibit defects in centrosome separation and spindle assembly (Cockell et al. 2004). Inactivation of both copies of LIS1 results in early embryonic lethality in the mouse (Hirotsune et al. 1998) as well as in Drosophila (Liu et al. 1999). In biochemical analysis, {alpha}2 has been shown to compete with NUDE and NUDEL for LIS1 binding (Kitagawa et al. 2000; Tarricone et al. 2004), suggesting that the presence of excess amount of the PAF-AH (I) catalytic subunit reduces binding of LIS1 to NUDE or NUDEL. Given the similarity of the centrosomal structure in cells over-expressing the catalytic subunit and in cells deficient in LIS1, our data supports the idea that the quantitative balance of {alpha}2 and LIS1 are crucial to LIS1 functions and that {alpha}2 acts as a negative regulator or a reservoir of LIS1 in the cytosol.

Phenotypical changes of {alpha}2 over-expressing cells are severer than those of {alpha}1 over-expressing cells. It is known that {alpha}2 has a higher affinity for LIS1 than does {alpha}l (Koizumi et al. 2003). In accordance with this, recombinant {alpha}1 has very low capacity of extracting LIS1 from the brain precipitate fraction (data not shown). It is thus reasonable to assume that LIS1 does not translocate too much extent from the centrosome and/or dynein complex fraction in {alpha}1 over-expressing cells. We have previously reported that a compositional change in the PAF-AH (I) catalytic subunits from {alpha}1/{alpha}2 to {alpha}2/{alpha}2 occurs during brain development (Manya et al. 1998). Expression of {alpha}1/{alpha}2 instead of {alpha}2/{alpha}2 may cause the distribution of LIS1 to shift from the cytosol to the centrosome and/or dynein complex fractions in actively migrating neurons.

The present results also demonstrate that substitution of Glu39 of the murine catalytic subunits by Asp or Ala resulted in a complete loss of LIS1 binding without reducing catalytic activity. In the {alpha}2 homodimer, the position of Glu39 is located at the opposite side of the catalytic center hole, suggesting that the LIS1 interacting site, containing Glu39, is distal to the catalytic center. Tarricone et al. (2004) have recently determined the crystal structure of the LIS1/({alpha}2/{alpha}2) complex. According to their data, LIS1 is a homodimer, forming a scissor-like structure via the N-terminal LisH domain (Kim et al. 2004), and binds symmetrically to one {alpha}2/{alpha}2 homodimer through the C-terminal WD repeats. They identified eleven residues involved in LIS1 binding in each {alpha}2 subunit. Five out of the eleven residues, including Glu39, are conserved in {alpha}1, but not in the Drosophila homologue, suggesting that these five residues are especially important for interaction with LIS1. We found that replacement of three out of these five residues in murine {alpha}2, namely Lys36, Asp37 and His181, with the corresponding residues in the Drosophila sequence did not alter their ability to bind LIS1 under the present assay conditions. Therefore, among the five residues, Glu39 is most important for the interaction between the catalytic subunits and LIS1. The amino acid in the Drosophila {alpha} homologue corresponding to Glu39 of mammalian {alpha} subunits is Asp. Glu and Asp both possess a carboxyl group in their side chain and differ only in the length of their carbon chain. Why this minor difference results in such a dramatic influence on LIS1 interaction is currently unknown. The side-chain carboxyl group of Glu39 forms a hydrogen bond with Arg238, Asn254 and Arg316 residues of LIS1 (Tarricone et al. 2004), while Asp may not be able to form such hydrogen bonds. In our hypothesis, interaction of Glu39 of the catalytic subunit with the three residues of LIS1 may induce a slight change in the surface structure of the catalytic subunit or LIS1 and facilitate the interaction of the other residues of the catalytic subunit with those of LIS1.

In contrast to the over-expression experiments, we have also performed RNAi-mediated knockdown of the PAF-AH (I) catalytic subunits in CHO cells and found that depletion of these proteins did not induce apparent phenotypic changes (data not shown). Taken together with the previous data that the catalytic subunit knockout mice ({alpha}1–/–/{alpha}2–/–) have no obvious abnormalities in tissues other than testis (Koizumi et al. 2003; Yan et al. 2003), these results suggest that the function of PAF-AH (I) catalytic subunits is not critical for most of cells under normal conditions. However, we could not exclude the possibility that subtle changes occur in PAF-AH (I) catalytic subunits-depleted cells.

In conclusion, we have demonstrated that over-expression of PAF-AH (I) catalytic subunits causes centrosomal amplification and pleiomorphic nuclei by interaction with endogenous LIS1, suggesting a link between over-expression of the catalytic subunits and cancerogenesis. Moreover, we have shown that single amino acid, namely Glu39 of the murine PAF-AH (I) catalytic subunit {alpha}2 is crucial for LIS1 binding. The {alpha}2-E39D mutant will be a useful tool for exploring the biological significance of the catalytic activity associated with the catalytic subunits of PAF-AH (I), since this mutant shows full catalytic activity but does not induce apparent cell abnormalities.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Gene construction and mutagenesis

Using mouse-brain RNA and reverse transcription-PCR (polymerase chain reaction), we generated full-length mouse {alpha}1, {alpha}2 and LIS1 cDNAs. The Drosophila PAF-AH (I) catalytic subunit homologue cDNA was a kind gift from Dr Zygmunt Derewenda (University of Virginia, Charlottesville, VA). To yield myc-tagged {alpha}2 and Drosophila homologue expression plasmids, {alpha}2 and Drosophila homologue cDNAs tagged with the myc epitope at the C-terminus were cloned into the pcDNA3 vector (Invitrogen, Carlsbad, CA). The LIS1 cDNA was inserted into pcDNA3 to form pcDNA3/LIS1. Mouse-Drosophila catalytic subunit chimeras and catalytic subunits harboring point mutations were generated by four-primer PCR (Ho et al. 1989). The amplified fragments were tagged with the myc epitope at the C-terminus and inserted into pcDNA3 vectors. To establish a tetracycline-regulated gene expression system (Tet-off), {alpha}1, {alpha}2, {alpha}2-S48C and {alpha}2-E39D cDNAs were cloned into pTRE expression vectors (Clontech, Palo Alto, CA), respectively.

Cell culture and transfections

CHO (Tokyo, Japan) cells were cultured in medium A [Ham's F12 medium (Nissui) supplemented with 2 mM glutamine, 10% fetal bovine serum, penicillin at 100 units/mL and streptomycin at 100 units/mL] at 37 °C in the presence of 5% CO2. CHO-AA8 cells stably transfected with the Tet-off transfection system were purchased from Clontech and maintained in medium A supplemented with G418 (Wako, Tokyo, Japan) at 100 µg/mL.

To establish CHO Tet-off cell lines, CHO-AA8 cells were transfected with the pTRE/{alpha}1, pTRE/{alpha}2, pTRE/{alpha}2-S48C or pTRE/{alpha}2-E39D using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. To facilitate clone selection, cells were co-transfected with a pTk-Hyg selection vector carrying a hygromycin B resistance gene (Clontech). After 48 h, stably transfected clones were selected by 100 µg/mL hygromycin B with 2 µg/mL tetracycline. Fourteen days after starting the culture in the hygromycin B-containing medium, {alpha}1-, {alpha}2-, {alpha}2-S48C- or {alpha}2-E39D-inducible clones were identified by Western blot analysis and immunofluorescence in the presence or absence of 2 µg/mL tetracycline.

Immunofluorescence

CHO cells grown on coverslips were washed briefly in phosphate-buffered saline (PBS) and fixed in 100% methanol at –20 °C for 10 min or in 3.7% paraformaldehyde in PBS for 10 min at room temperature. After paraformaldehyde fixation, cells were washed with PBS and permeabilized in 0.5% Triton X-100 in PBS for 10 min at room temperature. Cells were blocked with 3% BSA–PBS and incubated with primary or secondary antibodies in blocking buffer for 1 h at room temperature. Alexa 488-conjugated fluorescent secondary antibody (Molecular Probes, Eugene, OR) was used at a dilution of 1 : 2000 with 10 µg/mL 4', 6-diamino-2-phenylindole (DAPI, Sigma, St. Louis, MO). After incubation with antibodies, cells were washed 3 times in PBS for 5 min each at room temperature. Fluorescence microscopy was performed on a Axiophot 2 microscope (Carl Zeiss, Oberkochen, Germany).

Western blot analysis

Cell extracts were prepared by solubilization with cell lysis buffer containing 1% Triton X-100, 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% protease inhibitor cocktail (Sigma), 1% phosphatase inhibitor cocktail (Sigma) and centrifugation at 10 000 g for 20 min. The cell extracts and gel filtration fractions obtained were separated by SDS-PAGE, and the proteins were transferred to polyvinylidine difluoride (PVDF) membrane (Millipore, Bedford, MA). The following antibodies were used for Western blot analysis: anti-{alpha}-tubulin and anti-{gamma}-tubulin antibodies (Sigma); anti-{alpha}1 and anti-{alpha}2 antibodies (Koizumi et al. 2003); anti-LIS1 antibody (a kind gift from Dr Orly Reiner; Weizmann Institute, Israel); anti-NUDEL and anti-myc antibodies (Yamaguchi et al. 2004); and anti-p150Glued antibody (BD Biosciences, San Jose, CA). The immune reaction was developed by enhanced chemiluminescence kit (GE Healthcare Biosciences, Piscataway, NJ).

Immunoprecipitation

CHO cells were transiently co-transfected with pcDNA3/LIS1 and each of the myc-tagged catalytic subunit chimeras or control constructs were cultured for 48 h, lysed in cell lysis buffer, incubated for 20 min at 4 °C, and centrifuged at 10 000 g for 20 min at 4 °C. Samples were centrifuged at 10 000 g for 20 min at 4 °C. Supernatants were pre-incubated with protein G-Sepharose (GE Healthcare Biosciences) for 1 h at 4 °C in a final volume of 1 mL centrifuged at 4 °C for 3 min at 1000 g, and incubated with 5 µg of Protein G-Sepharose-coupled anti-myc antibody. After 1 h agitation at 4 °C, pellets were washed 3 times in cell lysis buffer, resuspended in SDS sample buffer and separated by SDS-PAGE. Western blot analysis was performed with the anti-myc or anti-LIS1 antibodies.

Preparation of glutathione S-transferase (GST) fusion proteins

To prepare GST fusion proteins, mouse {alpha}2 and {alpha}2-E39D cDNAs were cloned into a multi-cloning site downstream of the sequence for GST in pGEX4T-1 (GE Healthcare Biosciences). These plasmids were transformed into the JM109 strain of E. coli and induced with isopropyl-ß-D-thiogalactopyranoside to produce GST fusion proteins. The bacteria were suspended in PBS, vigorously sonicated, and centrifuged at 10 000 g for 20 min. The supernatant was applied to a glutathione bead column and eluted with elution buffer containing 50 mM Tris–HCl (pH 8.0), 120 mM NaCl and 10 mM glutathione. Purified GST fusion proteins were dialyzed against a dialysis buffer (PBS containing 2 mM EDTA and 1 mM DTT).

LIS1 extraction from mouse brain precipitate fraction

Adult mouse brain was homogenized in four volumes (w/v) of SET buffer containing 10 mM Tris–HCl (pH 7.4), 250 mM sucrose, 1 mM EDTA, 1% protease inhibitor cocktail (Sigma), 1% phosphatase inhibitor cocktail (Sigma) and centrifuged at 1000 g for 5 min at 4 °C to remove the solid material. The supernatant was centrifuged at 100 000 g for 1 h at 4 °C to form precipitate P1 and supernatant S1. P1 was homogenized in a volume of SET buffer equal to the volume of the supernatant, and an amount corresponding to 1.5 mg protein was mixed with 360 µg GST-{alpha}2 (wild-type or E39D mutant) or control GST, followed by incubation for 1 h at 4 °C and centrifugation at 100 000 g for 1 h at 4 °C to obtain precipitate P2 and supernatant S2. The S2 and P2 fractions were then subjected to Western blot analysis.

Measurement of PAF-AH activity

CHO cells were transiently transfected with each of the myc-tagged catalytic subunit chimeras and point mutants using Lipofectamine 2000, incubated for 48 h, harvested, sonicated in assay buffer containing 50 mM Tris–HCl (pH 7.4), 5 mM EDTA, 150 mM NaCl and centrifuged at 10 000 g for 20 min at 4 °C to yield a cytosolic supernatant. PAF-AH activity of the supernatant was measured as described previously (Hattori et al. 1993), using [3H]acetyl-platelet-activating factor as the substrate.


   Acknowledgements
 
This research was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the 21st Century COE Program, National Institute of Biomedical Innovation and PRESTO (Japan Science and technology Corporation).


   Footnotes
 
Communicated by: Kohei Miyazono

* Correspondence: E-mail: harai{at}mol.f.u-tokyo.ac.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Aumais, J.P., Tunstead, J.R., McNeil, R.S., Schaar, B.T., McConnell, S.K., Lin, S.H., Clark, G.D. & Yu-Lee, L.Y. (2001) NudC associates with Lis1 and the dynein motor at the leading pole of neurons. J. Neurosci. 21, RC187.[Abstract/Free Full Text]

Cahana, A., Escamez, T., Nowakowski, R.S., Hayes, N.L., Giacobini, M., von Holst, A., Shmueli, O., Sapir, T., McConnell, S.K., Wurst, W., Martinez, S. & Reiner, O. (2001) Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization. Proc. Natl. Acad. Sci. USA 98, 6429–6434.[Abstract/Free Full Text]

Cockell, M.M., Baumer, K. & Gonczy, P. (2004) lis-1 is required for dynein-dependent cell division processes in C. elegans embryos. J. Cell Sci. 117, 4571–4582.[Abstract/Free Full Text]

Dawe, A.L., Caldwell, K.A., Harris, P.M., Morris, N.R. & Caldwell, G.A. (2001) Evolutionarily conserved nuclear migration genes required for early embryonic development in Caenorhabditis elegans. Dev. Genes. Evol. 211, 434–441.[CrossRef][Medline]

Dobyns, W.B., Reiner, O., Carrozzo, R. & Ledbetter, D.H. (1993) Lissencephaly. A human brain malformation associated with deletion of the LIS1 gene located at chromosome 17p13. JAMA 270, 2838–2842.[Abstract]

Faulkner, N.E., Dujardin, D.L., Tai, C.Y., Vaughan, K.T., O’Connell, C.B., Wang, Y. & Vallee, R.B. (2000) A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function. Nat. Cell Biol. 2, 784–791.[CrossRef][Medline]

Feng, Y., Olson, E.C., Stukenberg, P.T., Flanagan, L.A., Kirschner, M.W. & Walsh, C.A. (2000) LIS1 regulates CNS lamination by interacting with mNudE, a central component of the centrosome. Neuron 28, 665–679.[CrossRef][Medline]

Geiser, J.R., Schott, E.J., Kingsbury, T.J., Cole, N.B., Totis, L.J., Bhattacharyya, G., He, L. & Hoyt, M.A. (1997) Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways. Mol. Biol. Cell 8, 1035–1050.[Abstract]

Hattori, M., Adachi, H., Aoki, J., Tsujimoto, M., Arai, H. & Inoue, K. (1995) Cloning and expression of a cDNA encoding the ß-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase. J. Biol. Chem. 270, 31345–31352.[Abstract/Free Full Text]

Hattori, M., Adachi, H., Tsujimoto, M., Arai, H. & Inoue, K. (1994a) Miller–Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase. Nature 370, 216–218. Erratum in: Nature 370, 391.

Hattori, M., Adachi, H., Tsujimoto, M., Arai, H. & Inoue, K. (1994b) The catalytic subunit of bovine brain platelet-activating factor acetylhydrolase is a novel type of serine esterase. J. Biol. Chem. 269, 23150–23155.[Abstract/Free Full Text]

Hattori, M., Arai, H. & Inoue, K. (1993) Purification and characterization of bovine brain platelet-activating factor acetylhydrolase. J. Biol. Chem. 268, 18748–18753.[Abstract/Free Full Text]

Hirotsune, S., Fleck, M.W., Gambello, M.J., Bix, G.J., Chen, A., Clark, G.D., Ledbetter, D.H., McBain, C.J. & Wynshaw-Boris, A. (1998) Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nat. Genet. 19, 333–339.[CrossRef][Medline]

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef][Medline]

Ho, Y.S., Swenson, L., Derewenda, U., Serre, L., Wei, Y., Dauter, Z., Hattori, M., Adachi, T., Aoki, J., Arai, H., Inoue, K. & Derewenda, Z.S. (1997) Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer. Nature 385, 89–93.[CrossRef][Medline]

Kawamura, K., Izumi, H., Ma, Z., Ikeda, R., Moriyama, M., Tanaka, T., Nojima, T., Levin, L.S., Fujikawa-Yamamoto, K., Suzuki, K. & Fukasawa, K. (2004) Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression. Cancer Res. 64, 4800–4809.[Abstract/Free Full Text]

Kim, M.H., Cooper, D.R., Oleksy, A., Devedjiev, Y., Derewenda, U., Reiner, O., Otlewski, J. & Derewenda, Z.S. (2004) The structure of the N-terminal domain of the product of the lissencephaly gene Lis1 and its functional implications. Structure (Camb) 12, 987–998.[Medline]

Kitagawa, M., Umezu, M., Aoki, J., Koizumi, H., Arai, H. & Inoue, K. (2000) Direct association of LIS1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE. FEBS Lett. 479, 57–62.[CrossRef][Medline]

Koizumi, H., Yamaguchi, N., Hattori, M., Ishikawa, T.O., Aoki, J., Taketo, M.M., Inoue, K. & Arai, H. (2003) Targeted disruption of intracellular type I platelet activating factor-acetylhydrolase catalytic subunits causes severe impairment in spermatogenesis. J. Biol. Chem. 278, 12489–12494.[Abstract/Free Full Text]

Lecointe, N., Meerabux, J., Ebihara, M., Hill, A. & Young, B.D. (1999) Molecular analysis of an unstable genomic region at chromosome band 11q23 reveals a disruption of the gene encoding the {alpha}2 subunit of platelet-activating factor acetylhydrolase (Pafah1a2) in human lymphoma. Oncogene 18, 2852–2859.[CrossRef][Medline]

Liu, Z., Xie, T. & Steward, R. (1999) Lis1, the Drosophila homolog of a human lissencephaly disease gene, is required for germline cell division and oocyte differentiation. Development 126, 4477–4488.[Abstract]

Manya, H., Aoki, J., Watanabe, M., Adachi, T., Asou, H., Inoue, Y., Arai, H. & Inoue, K. (1998) Switching of platelet-activating factor acetylhydrolase catalytic subunits in developing rat brain. J. Biol. Chem. 273, 18567–18572.[Abstract/Free Full Text]

Meerabux, J.M., Cotter, F.E., Kearney, L., Nizetic, D., Dhut, S., Gibbons, B., Lister, T.A. & Young, B.D. (1994) Molecular cloning of a novel 11q23 breakpoint associated with non-Hodgkin's lymphoma. Oncogene 9, 893–898.[Medline]

Niethammer, M., Smith, D.S., Ayala, R., Peng, J., Ko, J., Lee, M.S., Morabito, M. & Tsai, L.H. (2000) NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein. Neuron 28, 697–711.[CrossRef][Medline]

Reiner, O., Carrozzo, R., Shen, Y., Wehnert, M., Faustinella, F., Dobyns, W.B., Caskey, C.T. & Ledbetter, D.H. (1993) Isolation of a Miller–Dieker lissencephaly gene containing G protein ß-subunit-like repeats. Nature 364, 717–721.[CrossRef][Medline]

Sapir, T., Elbaum, M. & Reiner, O. (1997) Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit. EMBO J. 16, 6977–6984.[CrossRef][Medline]

Sasaki, S., Shionoya, A., Ishida, M., Gambello, M.J., Yingling, J., Wynshaw-Boris, A. & Hirotsune, S. (2000) A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system. Neuron 28, 681–696.[CrossRef][Medline]

Sheffield, P.J., Garrard, S., Caspi, M., Aoki, J., Arai, H., Derewenda, U., Inoue, K., Suter, B., Reiner, O. & Derewenda, Z.S. (2000) Homologs of the {alpha}- and ß-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome. Proteins 39, 1–8.[CrossRef][Medline]

Smith, D.S., Niethammer, M., Ayala, R., Zhou, Y., Gambello, M.J., Wynshaw-Boris, A. & Tsai, L.H. (2000) Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1. Nat. Cell Biol. 2, 767–775.[CrossRef][Medline]

Tarricone, C., Perrina, F., Monzani, S., Massimiliano, L., Kim, M.H., Derewenda, Z.S., Knapp, S., Tsai, L.H. & Musacchio, A. (2004) Coupling PAF signaling to dynein regulation: structure of LIS1 in complex with PAF-acetylhydrolase. Neuron 44, 809–821.[Medline]

Xiang, X., Osmani, A.H., Osmani, S.A., Xin, M. & Morris, N.R. (1995) NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. Mol. Biol. Cell 6, 297–310.[Abstract]

Yamaguchi, N., Takanezawa, Y., Koizumi, H., Umezu-Goto, M., Aoki, J. & Arai, H. (2004) Expression of NUDEL in manchette and its implication in spermatogenesis. FEBS Lett. 566, 71–76.[CrossRef][Medline]

Yan, W., Assadi, A.H., Wynshaw-Boris, A., Eichele, G., Matzuk, M.M. & Clark, G.D. (2003) Previously uncharacterized roles of platelet-activating factor acetylhydrolase 1b complex in mouse spermatogenesis. Proc. Natl. Acad. Sci. USA 100, 7189–7194.[Abstract/Free Full Text]

Received: 14 June 2007
Accepted: 5 July 2007





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