Report |
Address correspondence to Thomas M. Guadagno, H. Lee Moffitt Cancer Center & Research Institute, Building MRC 3 annex, 12902 Magnolia Drive, Tampa, FL 33612. Tel.: (813) 903-6818. Fax: (813) 903-6817. E-mail: guadagnt{at}moffitt.usf.edu
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Abstract |
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Key Words: MAP kinase; Rsk; spindle assembly; Xenopus; mitotic spindle
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Introduction |
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The Mos/MAP kinase kinase (Mek)/MAP kinase cascade is implicated in regulating the meiotic spindle during oocyte maturation (Verlhac et al., 1996). During the mitotic cell cycle, activation of MAP kinase (also known as extracellular signal-regulated kinase [ERK]) has been reported to regulate mitotic progression (Guadagno and Ferrell, 1998; Roberts et al., 2002) and microtubule dynamics (Gotoh et al., 1991; Guadagno and Ferrell, 1998). Consistent with a role in regulating the mitotic spindle, active forms of ERK1/2 are observed from prophase to anaphase at the spindle poles, spindle microtubules, kinetochores, and during cytokinesis at the midbody (Shapiro et al., 1998; Zecevic et al., 1998). Collectively, a picture has emerged suggesting the involvement of MAP kinase signaling in regulating the mitotic spindle. However, evidence directly supporting this possibility has been lacking.
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Results and discussion |
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Depletion of p42 MAP kinase leads to an increase in the length and polymerization of microtubules in Xenopus M phase egg extracts
Contrary to the static appearance of bipolar spindles, spindle microtubules are very dynamic with a turnover rate of 6090 s (Saxton et al., 1984). Therefore, we asked whether MAP kinase might play a role in regulating microtubule dynamics. To address this, we immunodepleted endogenous p42 MAP kinase (96%) from CSF-arrested egg extracts (Fig. 3 A) and measured the length and polymerization of microtubules using an aster assay. As evident in Fig. 3 B, microtubule asters were markedly larger in CSF-arrested extracts depleted of MAP kinase compared with mock-depleted extracts. Precisely, a 24% increase in mean aster radius was measured over three independent experiments (Table I). Furthermore, the average total fluorescence intensity/aster increased 30% in MAP kinasedepleted extracts compared with mock-depleted extracts, indicative of an increase in microtubule polymerization (Table I). Consistent with this, an increase in tubulin was observed in pelleted microtubules from CSF-arrested extracts depleted of MAP kinase activity (Fig. 3 C). Importantly, the addition of recombinant (his)6-tagged MAP kinase protein to depleted extracts restored MAP kinase activity (Fig. 3 A) and rescued the effects on microtubule polymerization and microtubule length (Fig. 3, B and C, and Table I). Together, our data support a role for MAP kinase in regulating microtubule dynamics. Further analysis will be required to precisely define which of the parameters of microtubule dynamics are regulated by MAP kinase.
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Since its early discovery as a microtubule-associated protein kinase (Ray and Sturgill, 1987; Reszka et al., 1995), MAP kinase has been suspected of regulating microtubules. Our data provide the first biochemical evidence that directly demonstrates a requirement for MAP kinase in the assembly and maintenance of the mitotic spindle. Our data also support a role for MAP kinase in regulating microtubule dynamics (Figs. 3 and 4). Furthermore, our Rsk immunodepletion data (Fig. 2) allow us to propose a model through which the Mek/MAP kinase/Rsk cascade bifurcates at MAP kinase and Rsk to elicit separate biological responses during mitosis (see model; Fig. 5 E). In this proposal MAP kinase likely targets spindle regulators (directly or indirectly) by phosphorylation to mediate spindle assembly and stability. Support for this comes from Verlhac and colleagues who have identified two MAP kinaseinteracting proteins, MISS (Lefebvre et al., 2002) and DOCR1 (Verlhac, M.-H., personal communication); both proteins are phosphorylated by MAP kinase and appear to be necessary for regulation of the meiotic spindle.
Contrary to our p90 Rsk immunodepletion results (Fig. 2), an active p90 Rsk mutant injected into progesterone-treated Xenopus oocytes incubated with U0126 was shown to restore both CSF arrest and spindle formation in the absence of MAP kinase activity (Gross et al., 2000). This discrepancy might reflect differences in the regulation of the meiotic spindle versus the mitotic spindle. Indeed, in our study sperm DNAassociated centrioles are supplemented in the Xenopus egg extracts to recapitulate the assembly of the mitotic spindle. Clearly, further studies are required to define the precise roles of MAP kinase and p90 Rsk during meiosis and mitosis. Nevertheless, our data show that depletion of both Rsk1 and Rsk2 has no effect on spindle assembly in the presence of active MAP kinase. Based on Rsk's role as an essential MAP kinase mediator for establishing (but not maintaining) the CSF arrest in unfertilized vertebrate oocytes (Bhatt and Ferrell, 1999; Gross et al., 2000), Rsk activation during mitosis may mediate cell cycle events associated with suppressing the metaphase-to-anaphase transition (Schwab et al., 2001). This implies that MAP kinase and Rsk cooperate in coordinating spindle assembly and mitotic progression.
The complex localization pattern of active MAP kinase at the mitotic spindle (Shapiro et al., 1998; Zecevic et al., 1998; Fig. 4 A) suggests multiple targets are under its regulation. As such, blocking MAP kinase activation would perturb the activity of many downstream components of this pathway. These components might include MISS or DOCR1, as well as conventional MAPs, microtubule-destabilizing proteins, and microtubule-based motor proteins. Consistent with this proposal, our studies show multiple spindle abnormalities in the absence of MAP kinase signaling, and this may represent a loss of function of several microtubule regulators. Studies are in progress to identify MAP kinase targets that are linked to its role in regulating the mitotic spindle. Although the regulation of the mitotic spindle by other signaling pathways (i.e., Ran, Cdc2, Plks, and Aurora) has been shown (Nigg, 2001), our data argue that the MAP kinase pathway is an important component of this signaling network.
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Materials and methods |
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Aster assays and microtubule pelleting
Microtubule polymerization was stimulated from sperm DNA (250/µl) in fresh CSF-arrested extracts (mock or MAP kinase depleted) for 10 min at 24°C in the presence of rhodamine-labeled tubulin. Microtubule asters were fixed in 0.25% glutaraldehyde solution containing BRB80 (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2, pH 6.8) and 0.5% Triton X-100 for 5 min at 24°C. Alternatively, microtubules were pelleted to assess total polymerized tubulin in extracts. Specifically, 20 µl of extract was diluted into 0.5 ml BRB80/30% glycerol/1% Triton X-100, layered onto a 1-ml BRB80/40% glycerol cushion, and centrifuged for 20 min, 14,000 rpm, at 4°C. The pellet was resuspended in SDS sample buffer, separated by 10% SDS PAGE, and immunoblotted for -tubulin. Quantification of
-tubulin bands was performed using Image Quant v5.0 software.
Recombinant protein production and purification
pGEX plasmids encoding GST fused to nondegradable sea urchin cyclin B1 (missing 13 NH2-terminal amino acids) or wild-type Xenopus MKP-1 (gift from Jim Ferrell and Mike Sohaskey [Stanford University, Stanford, CA]) were transformed into the bacterial strain BL21(DE3), grown in 2 liters LB media to an OD595 of 0.6 at 37°C, and induced for protein expression with 0.2 mM IPTG at 30°C for 3 h. Recombinant GST
cyclin B1 was purified from bacterial lysates as previously described (Solomon et al., 1990). Recombinant GST-MKP1 protein was affinity purified on glutathionesepharose beads as suggested (Amersham Biosciences). A plasmid encoding histidine (his)6-tagged Xenopus MAP kinase with a T7 promoter was transformed into BL21(DE3)pLysS, and recombinant fusion protein expressed in the presence of ampicillin (75 µg/ml) and chloramphenicol (35 µg/ml) using similar growth conditions as described above. Recombinant (his)6-tagged MAP kinase proteins were purified using talon metal affinity resin (CLONTECH Laboratories, Inc.). Eluted fractions containing recombinant GST- or (his)6-tagged fusion proteins were concentrated using Centricon 30 concentrators (Amicon Bioseparations) and buffer exchanged with XB buffer (10 mM Hepes, 1 mM MgCl2, 0.1 mM CaCl2, 100 mM KCl, 50 mM sucrose, pH 7.7, with KOH).
Immunodepletions
p42 MAP kinase was removed from CSF-arrested egg extracts by two rounds of immunodepletion as follows: protein Apurified anti-MAP kinase antibodies (X-15 serum kindly provided by Jim Ferrell) were prebound to 10-µl packed protein ASepharose 4B fast flow beads (Sigma-Aldrich), washed twice with 20 volumes of XB buffer, and incubated with 60 µl of fresh CSF-arrested egg extract for 4560 min on ice with occasional mixing. Then, the antibodybead complexes were pelleted for 15 s in an Eppendorf centrifuge. The MAP kinasedepleted extract was carefully removed and subjected to one more round of depletion. Rsk1 and Rsk2 depletions were performed sequentially using polyclonal rabbit Rsk1 and goat p90 Rsk2 antibodies (Santa Cruz Biotechnology, Inc.), respectively. Mock-depletions were performed using affinity-purified whole molecule antirabbit IgG (Sigma-Aldrich). To analyze Rsk proteins bound to the beads, the antibodybead complexes were washed three times with EB buffer (80 mM ß-glycerol phosphate, pH 7.3, 20 mM EGTA, 15 mM MgCl2) containing 0.1% Triton X-100, boiled in SDS-PAGE sample buffer, and subjected to gel electrophoresis.
Cell culture and cell synchronization
NIH 3T3 cells (obtained from American Type Culture Collection) were maintained in DMEM supplemented with 10% calf serum and 46 µg/ml gentamycin. Cells were synchronized at the G1/S boundary using a double-thymidine treatment method as described (Spector et al., 1998). The G1/S-synchronized cells were washed with DMEM and incubated in DMEM containing 10% calf serum to resume cycling into S, G2, and M phase. Cell synchronization was monitored by FACS analysis of DNA content (performed by H. Lee Moffitt Cancer Center Flow cytometry core facility). The mitotic index was determined hourly by analyzing at least 200 cells using a Nikon phasecontrast inverted microscope.
Immunostaining of mitotic spindles
Xenopus egg extract (25 µl) containing rhodamine-labeled spindles was diluted 1:10 in BRB80/0.5% Triton X-100/0.25% glutaraldehyde and fixed for 5 min at room temperature. The fixed spindles were layered on a 5-ml BRB80/20% glycerol cushion and centrifuged onto 12-mm glass coverslips in a HB-6 rotor at 6,000 RPM for 22 min at 4°C. Following the aspiration of the supernatant, the coverslips were gently rinsed twice with 5 ml of BRB80 buffer and postfixed with 20°C methanol for 5 min. Spindles were subjected to indirect immunofluorescence with antiphosphoMAP kinase antibodies (Cell Signaling) diluted 1:100 in Tris-buffered saline (10 mM Tris, pH 7.4, and 150 mM NaCl) containing 2% BSA and 0.1% Triton X-100. Phospho-MAP kinase staining was detected with Alexa Fluor 488 goat antirabbit IgG (Molecular Probes) secondary antibodies. DNA was stained with 1 µg/ml Hoechst in PBS. NIH 3T3 cells grown on glass coverslips were fixed in 4% paraformaldehyde (EM Sciences) at 4°C for 20 min, permeabilized for 1 h with 0.5% Triton X-100 in PBS, and labeled with anti-tubulin (Sigma-Aldrich, clone B-51-2) and Alexa Fluor 488 goat antimouse IgG secondary (Molecular Probes) antibodies to detect microtubule spindles. Antibodies were diluted in PBS/0.1% Triton X-100 containing 2% BSA and incubated for 1 h at room temperature. Three washes were performed with PBS/0.1% Triton X-100. Slides were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories) to stain chromosomes.
Fluorescence imaging
Fluorescent images were captured by a Roper coolsnap HQ CCD camera mounted on a Nikon E800 fluorescence microscope and controlled by Metamorph software v5.0r1 (Universal Imaging Corp.). Image processing was performed using Metamorph and Adobe Photoshop 6.0 software.
Immunoblotting analysis
NIH 3T3 cells grown on 100-mm dishes were collected in ice-cold 1X PBS, pelleted by centrifugation, and lysed in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, 50 mM sodium fluoride, 200 µM sodium vanadate, 40 mM ß-glycerophosphate) supplemented with fresh protease inhibitors (100 µM PMSF; 1 µg/ml each of leupeptin, pepstatin A, and aprotinin). Cell lysates were centrifuged for 10 min at 4°C at 14,000 g. Clarified supernatants were transferred to new tubes, and protein concentrations were determined by standard Bio-Rad Laboratories protein assays. 50 µg of protein from NIH 3T3 cell lysates or 25 µg of protein from Xenopus egg extracts was separated by 10% SDS-PAGE, electrotransferred onto Immobilon-P membranes, and examined by immunoblot analysis. Antibodies used include rabbit polyclonal antiXenopus MAP kinase peptide antibody X-15 or a similar one prepared in our laboratory, goat-polyclonal Rsk2 antibodies (Santa Cruz Biotechnology, Inc.), rabbit polyclonal Rsk1 antibodies (Santa Cruz Biotechnology, Inc.), p44/42 Phospho-MAP kinase monoclonal antibody (Cell Signaling), and phospho-JNK-1 (G-7) monoclonal (Santa Cruz Biotechnology, Inc.). A rabbit polyclonal Erk2 antibody (Transduction Laboratories) was used to detect total Erk protein in NIH 3T3 cell lysates. Species-specific alkaline phosphataseconjugated secondary IgG antibodies were obtained from Jackson ImmunoResearch Laboratories. Antibodies were diluted in 5% dry milk in PBS/0.1% Tween 20 and incubated for 1 h at room temperature or, in the case for p44/42 phospho-MAP kinase monoclonal antibody, incubated overnight at 4°C. To visualize protein bands, blots were incubated for 5 min with CDP-Star chemiluminescence substrate (Roche Diagnostic) and exposed to Kodak Biomax MS film.
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Acknowledgments |
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We acknowledge the support of the flow cytometry and analytical microscopy core facilities at the H. Lee Moffitt Cancer Center. This work was supported by the National Institutes of Health (grant GM62542) and a Moffitt institutional award from the American Cancer Society (to T.M. Guadagno). T.M. Guadagno was supported as a Special Fellow of the Leukemia & Lymphoma Society. M.M. Horne is a predoctoral fellow supported by the American Heart Association-Florida division.
Submitted: 25 April 2003
Revised: 14 May 2003
Accepted: 14 May 2003
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