From the
Center for Cell Signaling, University of
Virginia School of Medicine, Charlottesville, Virginia 22908 and
¶Department of Pharmacology and Cancer Biology,
Duke University Medical Center, Durham, North Carolina 27710
Received for publication, January 23, 2003 , and in revised form, March 13, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphorylation of I-2 in animal tissues has been studied. Depaoli-Roach
and Lee (8) immunoprecipitated
I-2 from 32P metabolically labeled mouse diaphragm. Phospho-amino
acid analysis showed that I-2 was phosphorylated on both serine and threonine,
with nine times more [32P]phosphoserine than
[32P]phosphothreonine. Holmes et al.
(9) purified I-2 from rabbit
skeletal muscle and using mass spectrometry showed that Ser86 (0.7
mol/mol), Ser120, and Ser121 (0.3 mol/mol each) were
phosphorylated, but no phosphorylation of Thr72 was detected.
Lawrence et al. (10)
immunoprecipitated I-2 from 32P-labeled mouse fat cells and showed
the presence of both [32P]phosphoserine and
[32P]phosphothreonine, with the phosphothreonine level being less
than 10% of phosphoserine. Moreover, despite the fact that GSK3 is inactivated
in response to insulin, threonine phosphorylation of I-2 was unchanged by
insulin treatment of the fat cells
(10). This suggested that
phosphorylation of Thr72 in vivo was most likely not
regulated by GSK3. Physiological regulation of Thr72
phosphorylation in I-2 has not been demonstrated.
To analyze the phosphorylation of I-2 in living cells we generated a phospho-site-specific antibody that uniquely recognized I-2 phosphorylated at Thr72. Using this antibody, we show here that I-2 is indeed phosphorylated on Thr72 in cells, and this modification is enhanced nearly 25-fold during mitosis. Using kinase inhibitors we tested whether Thr72 phosphorylation of I-2 in mitotic cells was catalyzed by GSK3 or cyclin-dependent protein kinases. Immunostaining showed that phosphorylation of I-2 at Thr72 in mitotic cells was concentrated at centrosomes. The data suggested that reversible phosphorylation of I-2 may control centrosome-associated PP1 activity, which could play a role in mammalian cell division.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Synchronization and Cell Lysate PreparationHeLa cells were synchronized by incubating with 400 µM mimosine for 24 h or with 200 ng/ml nocodazole for 16 h. Cells were rinsed with PBS and scraped into SDS-containing sample buffer.
Inhibitor 2 ImmunoprecipitationHeLa cells were rinsed with
room temperature PBS, and the dish was placed on a mixture of acetone and dry
ice, and 1 ml of ice-cold 5% trichloroacetic acid was added for 10 min. Cells
were scraped from the dish using a Costar cell lifter. The slurry was
transferred to a 1.7-ml microcentrifuge tube and centrifuged for 10 min at
10,000 x g. The supernatant was discarded, and the pellet was
washed twice with cold acetone and resuspended in urea resolubilization buffer
(9 M urea, 20 mM Tris, 23 mM glycine, 1
mM EDTA, and 10 mM -glycerophosphate). This
suspension was incubated at 30 °C for
1 h until the protein pellet
was dissolved. The solution was diluted 10-fold with immunoprecipitation
buffer (1% Nonidet P-40, 200 mM NaCl, 50 mM Tris, pH 8,
1 mM EDTA, 1 mM EGTA, 0.5% deoxycholate, 20
mM
-glycerophosphate, 1 mM sodium orthovanadate,
0.1% 2-mercaptoethanol, 0.4 mM Pefabloc, 1 µM
Microcystin-LR). After centrifugation for 10 min at 10,000 x g
the supernatant was used for I-2 immunoprecipitation. Briefly, the lysate was
incubated at 4 °C for 2 h with I-2 antibody coupled to protein G beads.
The beads were pelleted and washed with wash buffer (0.1% Nonidet P-40, 50
mM MOPS, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5
mM EDTA, 1 µM Microcystin-LR, 20 mM
-glycerophosphate, 1 mM sodium orthovanadate, 0.1%
2-mercaptoethanol) three times. The beads were resuspended in SDS-containing
sample buffer, and the proteins were resolved by SDS-PAGE.
Mass Spectrometric AnalysisThe protein band corresponding
to I-2 was excised from the gel and destained with 0.5 ml of 50% methanol/5%
acetic acid overnight at room temperature before dehydration in 200 µl of
acetonitrile and drying in a vacuum centrifuge. The proteins were reduced by
addition of 50 µl of 10 mM dithiothreitol and alkylated by
addition of 50 µl of 100 mM iodoacetamide (each for 30 min at
room temperature). The gel slices were dehydrated in 200 µl of
acetonitrile, hydrated in 200 µl of 100 mM ammonium bicarbonate,
and dehydrated again with 200 µl of acetonitrile. The dehydrated gel pieces
were again dried in a vacuum centrifuge and rehydrated in 50 µl of 20
ng/µl ice-cold, sequencing-grade modified porcine trypsin (Promega) for 5
min on ice. Excess trypsin was removed, and digestion was carried out
overnight at 37 °C. The peptides released by the digestion were collected
by successive extractions with 50 µl of 50 mM ammonium
bicarbonate and 50 µl of 50% acetonitrile/5% formic acid (2x),
combining the extracts in a siliconized 0.6-ml microcentrifuge tube rinsed
previously with ethanol, water, and ethanol. The combined extract was
concentrated in a vacuum centrifuge to 20 µl prior to analysis on a
Finnigan LCQ (ThermoQuest) ion-trap mass spectrometer with a Protana nanospray
ion source that was interfaced to a self-packed 8-cm x 75-µm inner
diameter Phenomenex Jupiter 10-µm C18 reverse-phase capillary column. The
peptides (0.5 µl) were injected and eluted from the column with an
acetonitrile/0.1 M acetic acid gradient (285% acetonitrile
in 30 min) at a flow rate of 0.25 µl min1. The
microspray ion source was set at 2.8 kV, and the data were analyzed using a
full data-dependent acquisition routine with the amino acid sequence acquired
in the four scans before the cycle repeats. This produced 500 tandem mass
spectrometry spectra of peptides ranging in abundance over several orders of
magnitude.
Antibody Production and PurificationThe synthetic peptide,
MKIDEPTPYH, with the threonine phosphorylated was conjugated to either
hemocyanin or thyroglobulin. Antisera were produced by subcutaneous injection
of this peptide into rabbits. The serum was precleared over unphosphorylated
recombinant I-2. The unbound fraction was incubated with I-2 phosphorylated by
both CKII and GSK3 and coupled to AffiGel-15 (Bio-Rad). After several
washes with PBS, the column was washed with 50 mM NaPO4,
pH 5.5. The antibody that remained bound to the phosphorylated I-2 was eluted
with 0.1 M glycine, pH 1.8 in 10% dioxane. The antibody solution
was neutralized and concentrated. This antibody is referred to as
phospho-Thr72
(9).
In Vitro Kinase Assay and InhibitorsHeLa cells were treated
with lysis buffer (1% (V/V) Nonidet P-40, 50 mM MOPS, pH 7.4, 5
mM MgCl2, 150 mM NaCl, 50 mM NaF,
20 mM -glycerophosphate, 1 mM Microcystin-LR, 0.4
mM Pefabloc SC, 0.1% (V/V) 2-mercaptoethanol). Cells were incubated
in lysis buffer on ice for 10 min and then homogenized by passage through an
insulin syringe. Reactions were set up in 25 mM MOPS, pH 7.4, 50
mM NaCl, 50 mM MgCl2, 0.1% Nonidet P-40, 0.1%
2-mercaptoethanol, 0.2 mM Microcystin-LR, 10 mM NaF, 4
mM
-glycerophosphate, 0.1 mM Pefabloc SC, 0.1
mM ATP with 5 µg of I-2 in 50 µl. Cell homogenate was added
to a final concentration of 50 µg/ml. Reactions were incubated at 30 °C
for 1 h and stopped by addition of SDS-containing sample buffer and boiled for
5 min.
ImmunocytochemistryHeLa cells were cultured on fibronectincoated coverslips. Coverslips were washed once with PBS followed by incubation in cold 10% trichloroacetic acid for 10 min. Cells were rinsed three times with PBS and permeabilized with 0.1% Triton-X-100 for 15 min. Specimens were blocked with 3% bovine serum albumin/1% ovalbumin and incubated with a purified sheep antibody to I-2, the rabbit antibody to phospho-Thr72 I-2, or both. A goat anti-sheep IgG antibody-conjugated rhodamine (Amersham Biosciences) or a goat anti-rabbit IgG conjugated to Alexafluor was used for immunofluorescent imaging. Coverslips were mounted onto glass slides with Vectorshield mounting medium (Vector Laboratories). Cells were visualized using a Nikon Microphot-5A microscope with x20 objective, and images were captured with a Hamamatsu C4742 digital camera operated by Open-Lab software (Improvision) and processed in Adobe Photoshop.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Phosphorylation of I-2 in Living CellsHeLa cells were prepared at different phases of the cell cycle, either unsynchronized (U, mostly interphase; see Fig. 2) or synchronized by addition of mimosine (to block at S phase) or nocodazole (to block at mitosis). Cell lysates were subjected to SDS-PAGE and compared by Western blotting (Fig. 2A). I-2 migrated as a single band in either unsynchronized or S-phase cells and was recognized by anti-I-2 antibody. This form of I-2 was not reactive with phospho-Thr72 antibody, suggesting that Thr72 in I-2 was predominantly unphosphorylated in these cells. However, the I-2 recovered from nocodazole-treated cells displayed an additional band of I-2 with decreased mobility. This lower mobility form of I-2 was readily recognized by the anti-phospho-Thr72 antibody, showing phosphorylation of I-2 at Thr72 in cells during mitosis.
|
We immunoblotted total cell proteins from unsynchronized and mitotic HeLa cells with the phospho-Thr72 site-specific antibody (Fig. 2B). In proteins from mitotic cells only one band corresponding to phospho-Thr72 I-2 was specifically stained near 35 kDa, and this was not detected in unsynchronized cells. One nonspecific band at 50 kDa was stained in both samples with equal intensity. These results further demonstrated the high degree of specificity of the phospho-Thr72 antibodies and confirmed that phosphorylation of I-2 at the Thr72 site occurred in particular during mitosis.
To directly determine the phosphorylation state of all the other sites in I-2 in unsynchronized HeLa cells, we employed the trichloroacetic acid fixation method. This protocol was first developed for immunohistochemistry, to preserve the phosphorylation state of proteins that were otherwise rapidly dephosphorylated (12). We adapted this method to purify phosphorylated I-2. Cells were directly treated with trichloroacetic acid, scraped, and centrifuged, and the trichloroacetic acid-precipitated proteins were resuspended in a saturated urea solution. Following the dilution of the urea solution, I-2 was immunoprecipitated, resolved by SDS-PAGE, and visualized by Coomassie blue staining. The I-2 protein was excised from the gel and digested with trypsin. Mass spectrometry revealed that Ser120 and Ser121 were phosphorylated to a stoichiometry of 0.9 mol/mol each. An additional phosphate was also detected in a peptide corresponding to residues 7090 (which contains a single serine residue, Ser86), but this could not be further analyzed because of low yield of this peptide (Fig. 3). Alkaline phosphatase treatment of HeLa cell lysates resulted in conversion of I-2 to a single band that migrated faster than all of the forms seen in Fig. 2A, consistent with Ser phosphorylation of I-2 in cells throughout the cell cycle (data not shown).
|
Thr72 Phosphorylation of I-2 during MitosisTo examine in more detail the phosphorylation of I-2 on Thr72 during mitosis, we employed immunoblotting with the phospho-Thr72 site-specific antibody using unsynchronized (U) and mitotic cells (M) (see Fig. 4) isolated by shaking off cells from nocodazole-treated cultures. The shake-off procedure greatly enriched the population of mitotic cells, relative to nocodazole treatment alone. Using approximately the same amount of total I-2 protein in the samples, immunoblotting with anti-phospho-Thr72 demonstrated a 25-fold increase in I-2 phosphorylation at Thr72 in mitotic HeLa cells (Fig. 4).
|
We prepared lysates from unsynchronized, mimosine-synchronized, or nocodazole-synchronized HeLa cells to determine whether increased I-2 (Thr72) phosphorylation during mitosis corresponded to an increase in kinase specific activity. These lysates were used as source of I-2 kinase activity. The same amount of lysate protein was incubated with recombinant I-2 plus MgATP. The results of the phosphorylation reactions were analyzed by Western blotting with the anti-phospho-Thr72 antibody (Fig. 5A). Unsynchronized cells displayed a low level of I-2 (Thr72) kinase activity. In contrast, little or no I-2 (Thr72) kinase activity was observed in lysates of mimosine-synchronized cells. Lysates from nocodazole-treated cells displayed significantly higher I-2 (Thr72) kinase activity, indicating that increased I-2 (Thr72) phosphorylation observed during mitosis resulted from the activation of a mitotic I-2 (Thr72) kinase.
|
Studies with purified protein kinases have suggested that GSK3 is the most effective I-2 (Thr72) kinase. To test whether GSK3 was the major I-2 kinase in mitotic cells we assayed I-2 phosphorylation by mitotic cell lysates in the presence of kinase inhibitors. Addition of a known GSK3 inhibitor (3-(3-carboxy-4-chloroanilino)-4-(3-nitrophenyl)maleimide) had no effect on the I-2 Thr72 kinase activity of the mitotic cell lysates (Fig. 5B). Another GSK3 inhibitor (2-thio(3-iodobenzyl)-5-(1-pyridyl)(1,3,4)-oxadiazole) likewise had no effect (not shown). In contrast, roscovitine, an inhibitor of cyclin-dependent protein kinases (CDK), decreased I-2 Thr72 phosphorylation by 60% (Fig. 5B). The results show that in mitotic cells phosphorylation of Thr72 in I-2 was not because of GSK3, but probably a CDK.
Analysis of I-2 (Thr72) Phosphorylation in CellsHeLa cells were stained using immunohistochemistry with the phospho-Thr72 site-specific I-2 antibody (Fig. 6A). We demonstrated the specificity of this antibody in this protocol, in addition to the results seen in Fig. 2. Pre-mixing the antibody with 1 µM phospho-Thr72 I-2 abolished all the immunostaining (Fig. 6C), in contrast with pre-mixing with 1 µM unphosphorylated I-2, which had little effect on staining (Fig. 6E). DNA was stained using DAPI (shown in Fig. 6, B, D, and F) to show the alignment of condensed chromosomes at the metaphase plate, establishing that the stained cells were at the same stage of mitosis. Staining of HeLa cells with an I-2 antibody (Fig. 7A) and with the phospho-Thr72 antibody (Fig. 7B) revealed the most intense staining for phospho-Thr72 occurred at the centrosomes. This is highlighted in the merged images of total plus phospho-I-2 (panel C) and in the phospho-Thr72 minus total I-2 images (panel D). Quantitation of the phospho-Thr72 staining relative to total I-2 revealed that I-2 (Thr72) phosphorylation peaked in prophase cells and decreased as cells progressed through mitosis (Fig. 7).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we produced a new experimental tool, namely an anti-phospho-Thr72 antibody, and showed I-2 becomes phosphorylated on Thr72 in living cells during mitosis. We also utilized gel mobility shifts and mass spectrometry to demonstrate that I-2 was phosphorylated throughout the cell cycle on three Ser residues. Our tests of kinase inhibitors indicate that GSK3, the best known I-2 kinase, contributes little to phosphorylation of I-2 during mitosis. Instead, the data point to other mitotic protein kinases. Roscovitine, a broad spectrum CDK inhibitor, blocked 60% of the I-2 (Thr72) kinase activity when used at a dose more than 10 times the IC50 for the purified cyclinB·cdc2 kinase complex. Thus, we conclude that one or more CDKs account for the majority of the mitotic I-2 (Thr72) kinase activity. Consistent with this finding, recent biochemical and genetic studies in yeast implicate PHO85, a cyclin-dependent protein kinase homologous to mammalian CDK5, as the sole kinase that phosphorylates Glc8 at the Thr118 site that is homologous to Thr72 (14). In addition, in HeLa cells there is another fraction of mitotic I-2 (Thr72) kinase that is resistant to roscovitine and as such is presumably distinct from the CDK family. Further work is required to establish the identity of this mitotic I-2 kinase.
Timely changes in phosphorylation of mitotic substrates require precise coordination of protein kinase and phosphatase activities. Genetic evidence with yeast, other fungi, and fruit flies points to a critical role for PP1 in various aspects of mitosis (1519). Inhibition of PP1 activity leads to premature mitotic entry (2022). In fungi and in mammals, PP1 activity appears particularly important for the metaphase-anaphase transition (16, 23, 24). However, the mechanisms that regulate PP1 activity during various stages of mitosis remain largely unknown.
Interestingly, we found phosphorylation of I-2 (Thr72) rose and fell during stages of mitosis with a peak at prophase. This suggests a highly dynamic regulation of I-2 and its associated PP1. These results are consistent with prior studies that showed PP1 activity is suppressed at entry into mitosis but required for the metaphase-anaphase transition (24). PP1 activity in mammalian cells also seems to be suppressed during telophase, to promote cell spreading (24). Thus, the cell cycle-dependent phosphorylation and dephosphorylation of I-2 on Thr72 may play a role in fluctuations in PP1 activity during the cell division cycle (2, 3, 16, 2023, 25). Measuring the activity of any one of the many forms of PP1 present in cells (associated with I-2 or not) is a daunting task that would require physical separation, as well as specific substrates.
The physiological function(s) of I-2 in mammalian cells is not well understood, and the presence of multiple pseudogenes has made gene deletion problematic. By demonstrating that I-2 (Thr72) phosphorylation at mitosis is highly focused at centrosomes, our data at least begin to point to a potential physiological role for this conserved modification. Our own recent evidence showed that I-2 is concentrated at centrosomes and associates in a complex with PP1 and the kinase Nek2. Overexpression of I-2 produced premature centrosome separation, consistent with activation of the Nek2 kinase (26). Nek2 is of specific interest, because it is localized at centrosomes, and prior studies have implicated a role for the NEK2·PP1 complex in the coordinate control of the phosphorylation of the associated substrate, cNAP (27). However, this is not the only target for I-2 action in cells, because I-2 also associates with neurabin/ spinophilin, as well as a novel transmembrane Ser/Thr kinase called KPI-2 (28, 29). Work of a decade ago (30) documented changes in the levels of I-2 during the cell cycle, with peak expression during mitosis, indicating a possible role for I-2 in mitosis. We speculate that phosphorylation of I-2 at Thr72 during mitosis plays a role in the control of centrosomes and maybe in other events at this critical stage of mammalian cell division.
![]() |
FOOTNOTES |
---|
Supported by Training Grant GF-10461 (to the University of Virginia Cell
and Molecular Biology Program).
|| To whom correspondence should be addressed: Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB7225, Charlottesville, VA 22908. Tel.: 434-924-5892; Fax: 434-243-2829; E-mail: db8g{at}virginia.edu.
1 The abbreviations used are: I-2, inhibitor-2; PP1, protein phosphatase 1;
GSK3, glycogen synthase kinase-3; CKII, casein kinase II; PBS,
phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; rI-2,
recombinant I-2; CDK, cyclin-dependent kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|