From the Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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GTP-binding protein/transglutaminases (tissue
transglutaminases or TGases) have been implicated in a variety of
cellular processes including retinoic acid (RA)-induced apoptosis.
Recently, we have shown that RA activates TGases as reflected by
stimulated GTP binding, increased membrane association, and stimulated
phosphoinositide lipid turnover. This prompted us to search for
cellular proteins that bind TGases in a RA-stimulated manner. In this
report, we show that the eukaryotic initiation factor (eIF-5A), a
protein that is essential for cell viability, perhaps through effects on protein synthesis and/or RNA export, associates with the TGase in vivo. The interaction between eIF-5A and TGase is
specific for the GDP-bound form of the TGase and is not detected when
the TGase is pre-loaded with GTPS. The TGase-eIF-5A interaction also is promoted by Ca2+, Mg2+, and RA treatment of
HeLa cells. In the presence of retinoic acid, millimolar levels of
Ca2+ are no longer required for the TGase-eIF-5A
interaction. Nocodazole treatment, which blocks the cell cycle at
mitosis (M phase), strongly inhibits the interaction between eIF-5A and
cytosolic TGase. The interaction between TGase and eIF-5A and its
sensitivity to the nucleotide-occupied state of the TGase provides a
potentially interesting connection between RA signaling and protein
synthesis and/or RNA trafficking activities.
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INTRODUCTION |
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Tissue transglutaminases
(TGases)1 represent an
interesting class of enzymes that were originally identified based on
their ability to catalyze the Ca2+-dependent
formation of covalent bonds between peptide-bound glutamyl residues and
the primary amino groups in a variety of compounds (1). It was then
realized that these enzymes are regulated by GTP and in fact can
undergo a GTP-binding/GTPase cycle like classical G proteins (2-4).
More recently, it also has been appreciated that these enzymes are
capable of ATP hydrolysis at a site distinct from the GTP-binding site
(5). The TGases appear to be widely distributed within the cell, mainly
present in the cytosol (6, 7), but are also found in plasma membranes
(8) and in the nucleus (9). They have been implicated in a variety of
fundamentally important cellular functions, including cell adhesion,
wound healing, and cellular differentiation and apoptosis (10-13). It
seems likely that the TGases play a role as signaling transducers in
some of these biological responses (again reminiscent of classical G
proteins), with one particular example being the mediation of
1-adrenergic receptor regulation of phosphoinositide lipid
turnover through the ability of the GTP-bound TGase to stimulate
phospholipase C-
1 (14).
Our laboratory has been interested in further probing the possible importance of the GTP-binding/GTPase cycle of TGases in their biological function. Recently, we found that retinoic acid (RA) treatment of HeLa cells under conditions that give rise to cellular differentiation and apoptosis strongly stimulated the GTP binding activity of the TGases (15). This was accompanied by an increased association of TGase with the plasma membrane and a concomitant stimulation of phospholipase C activity. These RA-stimulated effects could not be attributed to changes in the expression of the TGase, which caused us to propose that exposure of cells to RA may result in the expression of specific regulatory factors for the TGase, e.g. lipid-modifying enzymes and/or other proteins capable of imparting posttranslational modifications such as protein kinases.
In the present study, we set out to extend this work and identify possible regulatory factors as well as other potential target/effector molecules for the TGase. In particular, we followed up on an observation that an ~18-kDa protein appeared to be co-purifying with TGase from rabbit liver preparations. We have identified this protein as the eukaryotic initiation factor (eIF-5A), which was originally suspected to play an important role in protein synthesis but more recently has also been suggested to participate in nuclear (RNA) export (16). We show here that eIF-5A is a specific cellular binding partner for a particular form of the TGase and that this interaction is stimulated by RA, thus suggesting an interesting and potential link between protein synthesis and/or RNA trafficking and RA regulation of cellular activities.
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EXPERIMENTAL PROCEDURES |
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Construction of a Recombinant Baculovirus to Express Human Transglutaminase in Spodoptera frugiperda Sf21 Cells-- The cDNA clone of human tissue TGase was kindly provided by Dr. Peter Davies (University of Texas Medical School, Houston) in a pSG5 vector (Stratagene). It was digested with NcoI, and the resultant 2.2-kilobase fragment, including the coding region of the protein, was ligated into pGEXKG-lin (17), which was predigested with NcoI so that the TGase gene was fused in-frame downstream of the glutathione S-transferase (GST) coding region of the vector. The resultant construct was designated pGEXKG-lin-TG. The XbaI fragment of the pGEXKG-lin-TG containing the GST-tagged TGase was further subcloned into the baculovirus expression vector pVL1393 at the XbaI site.
The baculovirus expression vector containing GST-TGase was co-transfected with BaculoGold DNA into Sf21 cells using the BaculoGold transfection kit (Pharmingen, San Diego, CA). Recombinant viruses were isolated by plaque purification (18). Sf21 cells were infected by recombinant viruses at a multiplicity of infection of 5 plaque forming units/cell. The infected cells were harvested 60 h postinfection, and the cell pellets were stored frozen atProtein Purification of TGase from Sf21 Cells-- The pellets were thawed (50% w/v) in buffer A (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 100 µM phenylmethylsulfonyl fluoride and 10 µg/mL of leupeptin and aprotinin each) containing 1% Nonidet P-40 and incubated at 4 °C for 1 h. The suspension was centrifuged at 12,000 × g for 1 h, and the supernatant was mixed with glutathione-agarose beads (10% v/v) and incubated at 4 °C by continuous shaking for 1 h. The suspension was then centrifuged at 1000 × g for 10 min, and the beads were washed three times with buffer A and suspended in the same buffer (50% w/v) for storage.
Cell Culture-- HeLa cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml of penicillin and streptomycin, 2.0 g/liter sodium bicarbonate, and 10 mM HEPES in a humidified atmosphere with 5% (v/v) CO2 at 37 °C. To achieve cell-cycle arrest in M phase, nocodazole (80 ng/ml) was added to the growth medium, and cells were allowed to grow for 24 h. For harvesting, the cells were trypsinized and washed with RPMI 1640 containing 10% fetal calf serum.
Cell Fractionation-- Cells were pelleted and washed twice in ice-cold hypotonic buffer containing 10 mM HEPES, pH 8.0, 5 mM KCl, and 2 mM MgCl2. The cell pellets were then quick frozen in liquid nitrogen for storage. Thawed cells were resuspended in buffer B (25 mM HEPES, pH 7.4, 25 mM NaCl, 1 mM EDTA, 1 mM DTT, 100 µM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine, and 10 µg/ml aprotinin and leupeptin each). The suspension was homogenized using a Potter-Elvehjem homogenizer. Cellular debris was spun out at 3000 × g for 15 min. The supernatant was collected and spun at 100,000 × g for 45 min. Nonidet P-40 was added to the supernatant to a final concentration of 1% (this was used as a cytosolic fraction), and the membrane pellet was resuspended in 1% Nonidet P-40 in 25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM benzamidine, 100 µM phenylmethylsulfonyl fluoride, and 10 µg/mL aprotinin and leupeptin each.
Immunoprecipitations and Precipitation with
Glutathione-Agarose--
For the precipitation of eIF-5A by the
GST-TGase fusion protein, the clear supernatant representing the
cytosolic fraction of HeLa cells was transferred to fresh
microcentrifuge tubes. It was mixed with a suspension (25 µl) of
GST-TGase beads and incubated for 2 h at 4 °C in the presence
of different concentrations of salts (CaCl2 and
MgCl2) and nucleotides (GDP and GTPS) as indicated in
the figures. The suspension was centrifuged, and the pellet was washed
three times with buffer A and finally suspended in two times Laemmli
sample buffer (19) and boiled for 5 min. The supernatant was used for
the purpose of electrophoresis and Western blotting.
Identification of Proteins by Western Blot Analyses-- 20 µl of the immunoprecipitates were diluted with an equal volume of 2 × Laemmli sample buffer (19) and boiled for 5 min. The supernatants were subjected to electrophoresis on a 10% polyacrylamide gel with a 4% stacking gel. Proteins were transferred to 0.45 µM polyvinylidene fluoride filters (Millipore Corp.), and the filters were blocked using Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.4)/2% bovine serum albumin for 1 h. The blots were exposed to primary antibody against TGase in Tris-buffered saline, 0.2% Tween 20 (TTBS) or against eIF-5A in TTBS at a 1:250 dilution or a 1:500 dilution, respectively, and were washed three times at 15 min intervals. The blots were then exposed to secondary antibody (anti-mouse horseradish peroxidase or anti-rabbit horseradish peroxidase, Amersham Corp.) at a 1:5000 dilution in TTBS, 1% bovine serum albumin for 1 h, then washed three times with TTBS and two times with Tris-buffered saline, and visualized using a Chemiluminescence system (ECL, Amersham Corp.).
Photoaffinity Labeling of GTP-binding
Proteins--
Photoaffinity labeling of GTP-binding proteins with
[-32P]GTP was carried out as described previously (9).
Samples (20 µl) were incubated with 5 mM EDTA and 2-3
µCi [
-32P]GTP (0.02 µM) (20 µl final
volume) in tissue culture plates (96 wells) for 10 min at room
temperature in a reaction buffer that contained 50 mM
Tris-HCl, pH 7.4, 2 mM EGTA, 1 mM DTT, 20% (v/v) glycerol, 100 mM NaCl, and 500 µM
AMP(PNP). The samples were then placed in an ice bath, irradiated with
UV light (254 nm) for 15 min, mixed with 5 × sample buffer (19),
and boiled for 5 min. SDS-PAGE was performed (10% polyacrylamide), and
then the gels were stained, dried, and exposed on Kodak X-OMAT XAR-5 film using a DuPont image intensifier screen.
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RESULTS AND DISCUSSION |
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Various lines of evidence have suggested that GTP-binding protein/transglutaminases (TGases) may represent a new family of transducer molecules that upon binding GTP can stimulate target/effector activities such as phosphoinositide lipid turnover (8, 20). We recently reported that a number of biochemical activities of TGases are stimulated by RA including GTP binding (and the resultant GTP hydrolytic activity), transglutaminase activity, plasma membrane-association and phosphoinositide lipid turnover (15). The regulatory mechanisms underlying these RA-stimulated activities are not known, although they cannot be attributed to simple changes in the levels of TGase expression (15). As a means to gain further insight into the interplay between RA and TGases, as well as into the function of TGases, we have set out to identify potential regulators and other binding partners or targets for these GTP-binding proteins.
Based on an earlier observation that an ~18-kDa protein appeared to co-purify with TGases from rabbit liver,2 we obtained sufficient amounts of the 18-kDa protein for microsequence analysis and determined that it was the eukaryotic initiation factor-5A (eIF-5A). At present, little is known regarding the cellular functions of eIF-5A. It has been suggested to participate in some aspect of protein synthesis at the ribosomal subunit-joining step and/or at a later stage of 80 S ribosomal initiation complex formation (21). Recently, eIF-5A also has been suggested to play a role in RNA export, based on its interaction with the HIV-1 protein, Rev (16). The eIF-5A protein is unique in that it is the only cellular protein known to contain the unusual amino acid hypusine; a number of lines of evidence indicate that hypusine and eIF-5A are essential for eukaryotic cell viability (22). Likewise, there are a number of indications for an important involvement of TGases in cell cycle progression and cell growth regulation (6, 13, 23-26). Thus, a TGase-eIF-5A interaction would have interesting implications, particularly regarding the possible connection of RNA metabolism to growth regulation (27).
We first set out to determine whether we could obtain biochemical
evidence that eIF-5A is in fact a binding partner for TGase. To do
this, we expressed the TGase in insect cells as a GST fusion protein to
use this as an affinity reagent for identifying eIF-5A as a possible
target. The results presented in Fig. 1
(top panel) show that when the GST-TGase protein was
incubated with lysates from HeLa cells, the endogenous eIF-5A (as
detected by Western blotting with an anti-eIF-5A antibody) was
co-precipitated with GST-TGase upon the addition of
glutathione-agarose. This interaction appeared to be specific for the
GDP-bound form of the GTP-binding protein, since we could not detect an
interaction with eIF-5A when the GST-TGase was preloaded with GTPS.
The GST-TGase-eIF-5A interaction also required both Ca2+
and Mg2+ (Fig. 1, lower panel; also see
below).
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We next examined whether the TGase-eIF-5A interaction occurred in
intact cells. Using an anti-eIF-5A polyclonal antibody that can
immunoprecipitate eIF-5A from cells (Fig.
2, top panel), we found that
TGase was specifically co-precipitated with eIF-5A (Fig. 2,
middle panel, lanes 3 and 4) but not
with nonimmune IgG (Fig. 2, middle panel, lanes 1 and 2) as indicated by Western blotting with an anti-TGase
antibody. The TGase that co-precipitates with eIF-5A can also be
photoaffinity labeled with [-32P]GTP (Fig. 2,
lower panel, lanes 3 and 4).
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The ability of the eIF-5A-associated TGase to bind GTP indicates that
it belongs to an activated pool of the endogenous TGase. Previously, we
had shown that in HeLa cells most of the TGase was inactive and not
capable of binding [-32P]GTP (15). However, as alluded
to earlier, RA, which causes the cells to differentiate and undergo
apoptosis (13), strongly stimulated the GTP binding activity of the
cellular TGase (15). Since this did not appear to be due to a change in
the expression of the TGase, we proposed that RA treatment of cells
increased the expression of a lipid-modifying enzyme and/or possibly
another regulatory factor (e.g. a protein kinase) that
modified the TGase, thus creating an activated pool of protein that was
capable of GTP binding activity and an increased ability to associate
with the plasma membrane. Given this suggestion, we therefore reasoned that if it was the activated pool of TGase that associates with eIF-5A,
then RA treatment of cells should stimulate the formation of a
TGase·eIF-5A complex. The results presented in Fig.
3 show that this in fact is the case.
Specifically, following RA treatment of HeLa cells, TGase was detected
in co-immunoprecipitates with eIF-5A even after only a very brief
exposure (~2 min) of the blot and under conditions where in the
absence of RA, little detectable TGase was present in the eIF-5A
precipitates. Fig. 3 (right panel) also shows that RA
treatment strongly increased the amount of [
-32P]GTP
binding activity (corresponding to the TGase) that
co-immunoprecipitated with eIF-5A.
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We have been able to further probe the connection between RA-promoted
activation of TGase and its interaction with eIF-5A by taking advantage
of a recent finding that nocodazole treatment of HeLa cells (under
conditions that cause the cells to accumulate in M phase) (28) inhibits
the ability of membrane-associated TGase to bind GTP and thus uncouples
the ability of the RA-regulated TGase to associate with plasma
membranes from its ability to become activated (data not shown). It is
interesting that nocodazole treatment also strongly inhibits the
association of the cytosolic TGase with eIF-5A. The results presented
in Fig. 4 clearly show that the
co-precipitation of TGase with eIF-5A does not occur in HeLa cells that
have been pretreated with nocodazole, either when monitoring this
interaction by Western blotting with the specific anti-TGase antibody
(upper panel) or by photoaffinity labeling with
[-32P]GTP (lower panel). A similar
inhibitory effect by nocodazole was also obtained with RA-treated HeLa
cells (data not shown)
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The results presented in Fig. 5 show that the effects of both RA and nocodazole treatment are directed at eIF-5A as well as at the membrane-bound pool of TGase. Given that Ca2+ appeared to serve as a necessary cofactor for TGase-eIF-5A interactions (see Fig. 1), we performed an experiment to determine whether treatment with RA or nocodazole influenced the ability of Ca2+ to stimulate these interactions. Specifically, lysates were prepared from control HeLa cells and from RA-treated cells or nocodazole-treated cells and then incubated with the recombinant GST-TGase in the presence of different levels of CaCl2. The GST-TGase·eIF-5A complexes were precipitated with glutathione-agarose and Western blotted with the anti-eIF-5A polyclonal antibody. As shown in Fig. 5, the GST-TGase-eIF-5A interaction was maximal when lysates from control cells were incubated with 1 mM CaCl2, whereas in RA-treated cells GST-TGase-eIF-5A interactions were clearly detected even in the absence of added CaCl2. These results suggest that RA treatment has a direct effect on eIF-5A, such that it is able to associate with the TGase even at low (micromolar) levels of Ca2+. It should be noted that nocodazole treatment completely eliminated the interaction between GST-TGase and eIF-5A. Because nocodazole did not decrease the levels of eIF-5A detected in these lysates by Western blotting (data not shown), these findings suggest that the nocodazole effects on TGase-eIF-5A interactions are directed at eIF-5A, such that it cannot bind to the TGase even in the presence of millimolar levels of Ca2+. Thus, like RA, nocodazole exhibits regulatory effects on both partners of the TGase-eIF-5A binding interaction.
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These results when taken together with previous findings are consistent
with the following model (Fig. 6).
Treatment of HeLa cells with RA promotes the formation of an activated
pool of the TGase, which has an increased affinity for the plasma
membrane and the capability to undergo GTP/GDP exchange. That portion
of the membrane-associated TGase that binds GTP can then stimulate the
activity of a phospholipase C enzyme (15); recent work would suggest
that this is the phospholipase-1 isoform (14). The TGase-promoted
stimulation of phospholipase C activity gives rise to an increase in
intracellular Ca2+ which has been suggested to stimulate
the enzymatic tranglutaminase activity of the TGase (29). If
Ca2+ binding to the TGase has some type of regulatory
affect on the tranglutaminase active site, such an effect might account
for the ability of Ca2+ to promote TGase-eIF-5A
interactions. The involvement of the transglutaminase active site in
binding eIF-5A also might explain an earlier observation that the
unique hypusine residue of eIF-5A serves as substrate for
transglutaminase reactions, i.e. it can be cross-linked to
the
-carboxamide group of glutamine side chains (30). Within this
scheme, RA could promote TGase-eIF-5A interactions both by initiating a
cascade of events that results in the membrane association of TGase and
a resultant stimulation of phospholipase C activity and increase in
intracellular Ca2+, and by having an as yet undetermined
effect on the eIF-5A molecule (perhaps by influencing some type of
posttranslational modification), such that eIF-5A can bind the TGase at
micromolar levels of Ca2+. Nocodazole treatment by
inhibiting the activation of membrane-associated TGase, as well
as by exerting a regulatory effect on eIF-5A, inhibits TGase-eIF-5A
interactions.
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Clearly, a critical question concerns how the TGase-eIF-5A interaction influences the normal functions of these proteins. We have not found any affect on the various measurable TGase activities; however, as alluded to earlier, an interesting possibility is that TGases influence the cellular localization of eIF-5A. When the TGase is in a GDP-bound state (following GTP hydrolysis (31)), it is able to bind eIF-5A and maintain eIF-5A in the cytoplasm. However, upon GTP-GDP exchange, the eIF-5A is released perhaps allowing the molecule to bind to some other cytosolic (target) protein involved in protein synthesis and/or to return to the nucleus to participate in some aspect of RNA trafficking. Thus, the GTP-binding/GTPase cycle of the TGase could serve as a timing device to regulate changes in the cellular localization of eIF-5A that are important for either protein synthesis or RNA trafficking between the cytoplasm and the nucleus. The fact that nocodazole treatment perturbs both the GTP-binding/GTPase cycle of membrane-associated TGase, and the ability of eIF-5A to associate with the TGase, further suggests that eIF-5A activity might be coupled to the cell cycle. Taken together, these results would suggest that other factors that influence cell-cycle progression may also exert regulatory effects on TGase-eIF-5A interactions. Future studies will be directed toward testing the different possibilities raised above and determining the identity of additional cellular factors that mediate the regulation of TGase activities by RA as well as additional binding partners for eIF-5A.
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ACKNOWLEDGEMENTS |
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We thank Cindy Westmiller for her expert technical assistance in the preparation of this manuscript. We also thank Dr. Edith Wolff for the antibody against eIF-5A.
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FOOTNOTES |
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* This work was supported by Grants GM40654 and EY06429 from the National Institutes of Health, Dept. of Defense Grant DAMD17-94-J-4123, and by the Council for Tobacco Research Grant 4014.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The abbreviations used are: TGases,
transglutaminases; GTPS, guanosine
5
-3-O-(thio)triphosphate; RA, retinoic acid; eIF-5A, eukaryotic initiation factor-5A; GST, glutathione
S-transferase; DTT, dithiothreitol; TTBS, Tris-buffered
saline, 0.2% Tween 20; PAGE, polyacrylamide gel electrophoresis.
2 U. S. Singh, unpublished results.
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REFERENCES |
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