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• W2020921289 abstract "Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G2/M progression. The MAPK cascade has well established roles in entry and exit from mitosis inXenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G2/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr286. These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis. Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G2/M progression. The MAPK cascade has well established roles in entry and exit from mitosis inXenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G2/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr286. These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis. mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase phospho-MEK extracellular signal-regulated kinase phospho-ERK hemagglutinin green fluorescent protein phosphate-buffered saline glutathione S-transferase Ras-binding domain 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Growth factors signal cells to proliferate via a complex mechanism that links receptor activation to the cell cycle machinery. Receptor activation induces cells to progress from quiescence (G0) into G1 phase. Constant receptor stimulation is required until the restriction point is reached in mid-to-late G1. Once the restriction point is passed, cell cycle progression is regulated autonomously by the cell cycle machinery (1Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar). The primary components of this cell cycle machinery are a family of serine/threonine kinases, the cyclin-dependent kinases. Cyclin-dependent kinases are regulated by association with regulatory cyclin subunits and by phosphorylation (2Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). Passage through the eukaryotic cell cycle requires successive activation of different complexes between cyclin and cyclin-dependent kinase complexes. Progression from G2 to M phase is controlled by cyclin B-Cdc2. The MAPK1 cascade, controlled by the small GTPase Ras, is critical for progression from G1 to S phase (3Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar). Growth factor receptors recruit Ras guanine nucleotide exchange factors to the plasma membrane to activate Ras, which then recruits and activates Raf kinases. In turn, activated Raf phosphorylates and activates the dual-specificity MAPK kinases (MEK1 and MEK2) that phosphorylate and activate the MAPKs ERK1 and ERK2. Activated ERK phosphorylates a wide range of effector proteins, including transcription factors that increase expression of proteins required for cell cycle progression into S phase (3Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar). The MAPK pathway has a clearly defined role in G1 phase progression, but may also have a role in the G2/M phase of the cell cycle. In cycling Xenopus egg extracts, addition of activated MEK during interphase blocks cyclin B-Cdc2 activation by maintaining Wee1 (4Bitangcol J.C. Chau A.S. Stadnick E. Lohka M.J. Dicken B. Shibuya E.K. Mol. Biol. Cell. 1998; 9: 451-467Crossref PubMed Scopus (61) Google Scholar, 5Walter S.A. Guadagno S.N. Ferrell Jr., J.E. Mol. Biol. Cell. 2000; 11: 887-896Crossref PubMed Scopus (51) Google Scholar). ERK activity is dispensable for normal mitotic progression inXenopus, although ERK activity is required for the proper functioning of the mitotic spindle (6Minshull J. Sun H. Tonks N.K. Murray A.W. Cell. 1994; 79: 475-486Abstract Full Text PDF PubMed Scopus (352) Google Scholar). The role of the MAPK pathway in mammalian mitosis is less clear. The biochemical activities of endogenous ERK1 and ERK2 have been shown to decrease as cells enter mitosis (7Tamemoto H. Kadowaki T. Tobe K. Ueki K. Izumi T. Chatani Y. Kohno M. Kasuga M. Yazaki Y. Akanuma Y. J. Biol. Chem. 1992; 267: 20293-20297Abstract Full Text PDF PubMed Google Scholar, 8Edelmann H.M. Kuhne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), yet other studies conclude that activation of the MEK/ERK cascade is required for normal progression into mitosis (9Wright J.H. Munar E. Jameson D.R. Andreassen P.R. Margolis R.L. Seger R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11335-11340Crossref PubMed Scopus (159) Google Scholar, 10Hayne C. Tzivion G. Luo Z. J. Biol. Chem. 2000; 275: 31876-31882Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Components of the MAPK pathway have been shown to associate with the mitotic apparatus of mammalian cells. Activated MEK and ERK localize to the mitotic spindle from prophase to anaphase and to the midbody during cytokinesis (11Shapiro P.S. Vaisberg E. Hunt A.J. Tolwinski N.S. Whalen A.M. McIntosh J.R. Ahn N.G. J. Cell Biol. 1998; 142: 1533-1545Crossref PubMed Scopus (194) Google Scholar). Activated ERK also colocalizes with the kinetochore motor protein CENP-E, raising the possibility that CENP-E is a downstream effector for ERK during mitosis (12Zecevic M. Catling A.D. Eblen S.T. Renzi L. Hittle J.C. Yen T.J. Gorbsky G.J. Weber M.J. J. Cell Biol. 1998; 142: 1547-1558Crossref PubMed Scopus (194) Google Scholar). Blocking MEK activity in cycling somatic cells does not significantly affect mitotic entry, but it does slow progression through mitosis, probably by slowing the CENP-E-dependent chromosome movement coordinated by the mitotic spindle (13Roberts E.C. Shapiro P.S. Nahreini T.S. Pages G. Pouyssegur J. Ahn N.G. Mol. Cell. Biol. 2002; 22: 7226-7241Crossref PubMed Scopus (124) Google Scholar). A number of groups have investigated the regulation of Raf-1 in mitosis using ectopic expression of Raf-1 and nocodazole treatment to produce a mitotic checkpoint-arrested cell population. However, nocodazole treatment itself causes a rapid activation of Raf-1 independent of mitosis (10Hayne C. Tzivion G. Luo Z. J. Biol. Chem. 2000; 275: 31876-31882Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), probably due to disruption of the microtubule network (14Blagosklonny M.V. Schulte T.W. Nguyen P. Mimnaugh E.G. Trepel J. Neckers L. Cancer Res. 1995; 55: 4623-4626PubMed Google Scholar). Thus, little is actually known about the activity of the endogenous Ras/Raf/MEK/ERK pathway in normal G2/M progression. In this study, we examined the expression and activation of all components of the Ras/Raf/MEK/ERK pathway during G2/M progression. To avoid the use of microtubule-disrupting drugs like nocodazole that directly influence Raf activity independent of the cell cycle, HeLa cells were synchronized at G1/S and allowed to progress through mitosis in the absence of drugs. Endogenous proteins of the MAPK cascade were then analyzed biochemically. This approach excludes potentially confounding results arising from nocodazole treatment and ectopic protein expression. We show that membrane-bound MEK1 is strongly activated as cells transit into mitosis, but that this activation is uncoupled from ERK, which is inactive in mitosis. The uncoupling of MEK activation from ERK is mediated by direct modifications to MEK1 by the mitotic cell cycle machinery and requires active cyclin B-Cdc2. Roscovitine was purchased from BIOMOL Research Labs Inc. Compound 5 was a kind gift from Professor John Lazo (University of Pittsburgh). 4,6-Diamidino-2-phenylindole (dihydrochloride, hydrate) was purchased from Sigma. Rabbit anti-N-Ras, anti-K-Ras, anti-H-Ras, anti-B-Raf, anti-MEK1C, anti-MEK1N, anti-MEK2C, anti-MEK2N, anti-ERK1, and anti-ERK2 polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-phospho-MEK, anti-MEK1/2, and anti-phosphothreonine/proline polyclonal antibodies and anti-HA, anti-Raf-1, and anti-phospho-ERK antibodies were purchased from Cell Signaling Technology. Rabbit anti-cyclin B1 and anti-Cdc2 polyclonal antibodies were as previously described (15Wang X.Q. Gabrielli B.G. Milligan A. Dickinson J.L. Antalis T.M. Ellem K.A. Cancer Res. 1996; 56: 2510-2514PubMed Google Scholar). Mouse anti-α-tubulin monoclonal antibody was purchased from Amersham Biosciences. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse and fluorescein isothiocyanate-conjugated anti-mouse goat polyclonal antibodies were purchased from Zymed Laboratories Inc. Plasmid pLNCAL-HA-tagged wild-type MEK1, MEK1(S218D/S222D), MEK1Δ32–51, and MEK1(T286A) were a generous gift from Dr. Andy Catling (16Catling A.D. Schaeffer H.J. Reuter C.W. Reddy G.R. Weber M.J. Mol. Cell. Biol. 1995; 15: 5214-5225Crossref PubMed Scopus (167) Google Scholar). GFP- and HA-tagged MEK1ΔN51 was produced by PCR of the open reading frame of wild-type MEK1 or MEK1(S218D/S222D) from residue 51 to the termination codon, such that it was cloned in-frame into the pEGFP vector (Clontech). HeLa and 293H (Invitrogen) cells were grown in Dulbecco's modified Eagle's medium supplemented with 107 bovine donor serum (Serum Supreme, BioWhittaker, Inc.). Assays for Mycoplasma were carried out monthly to ensure that the cultured cells were free of contamination. HeLa cells were synchronized using a double thymidine block release protocol as described (17Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). Mitotic shake-off was used to obtain a highly enriched mitotic cell population from double thymidine-synchronized cells passing through mitosis. 293H cells were transfected using LipofectAMINE 2000 (Invitrogen) as directed by the manufacturer. HeLa cells were transfected by electroporation. Cell cycle status of cultures was assessed by flow cytometry as described previously (17Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). Floating and attached cells were harvested in ice-cold PBS and pelleted by low speed centrifugation (1000 rpm) at 4 °C. PBS was removed, and cell pellets were resuspended in 0.5 ml of buffer A (10 mm Tris-HCl (pH 7.5), 25 mm NaF, 5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, and 100 ॖm NaVO4) and homogenized by passage through a 23-gauge needle. The post-nuclear supernatants were spun at 100,000 × g; the supernatants (S100) were removed; and the sedimented fractions (P100) were rinsed and sonicated for 5 min in 100 ॖl of ice-cold buffer A. Protein content was measured by the Bradford reaction. P100 and S100 aliquots were snap-frozen and stored at −70 °C. Protein expression levels were determined by quantitative immunoblotting. 20 ॖg of P100 and an equivalent volume of S100 were resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes by semidry transfer. Proteins were detected using the appropriate primary antibodies followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody. Bands were visualized by enhanced chemiluminescence (ECL), and relative luminescence was quantified by phosphorimaging (Bio-Rad). Levels of Ras-GTP were assayed using bacterially expressed GST-Raf-RBD(K85A) to bind Ras-GTP, which was detected by immunoblotting with Ras isoform-specific antibodies (18Clyde-Smith J. Silins G. Gartside M. Grimmond S. Etheridge M. Apolloni A. Hayward N. Hancock J.F. J. Biol. Chem. 2000; 275: 32260-32267Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Membrane-bound MEK1 was solubilized for two-dimensional analysis by addition of 100 ॖl of 0.57 SDS in PBS to a 17 Nonidet P-40 membrane pellet (from a 10-cm dish of mitotic HeLa cells) and incubation at 37 °C for 10 min with vigorous shaking and then addition of 150 ॖl of 57 Nonidet P-40 and sonication for 10 min at 4 °C in a sonicating water bath. The solubilized protein was acetone-precipitated and resuspended in 250 ॖl of rehydration buffer (5 m urea, 2 mthiourea, 27 CHAPS, 27 SB310, 4 mm Tris-Cl (pH 9.5), and bromphenol blue to color) with freshly added dithiothreitol and pH 3–10 carrier ampholytes (two-dimensional) to final concentrations of 50 mm and 17, respectively. The first dimension was resolved on a pH 3–10 linear ImmobilineTM DryStrip (Amersham Biosciences) for 100 kV-h after 18 h of rehydration. The second dimension was resolved on 127 SDS-polyacrylamide gel. Western transfer and immunoblotting were performed as described above. Raf kinase assays were performed essentially as described (19Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, equivalent P100 aliquots (20 ॖg of total protein) were incubated in kinase buffer (buffer A containing 0.5 mm ATP and 5 mm MgCl2) at 30 °C for 30 min with recombinant MEK and ERK (tube A; to measure total Raf, MEK, ERK, and nonspecific kinase activities), ERK alone (tube B; to measure MEK, ERK, and nonspecific kinase activities), or buffer alone (tube C; to measure ERK and nonspecific kinase activities). Primary reactions were diluted in buffer A, and an aliquot was incubated in a second reaction containing [γ-32P]ATP and myelin basic protein. The radioactivity incorporated into myelin basic protein was quantified by phosphorimaging. Total P100 Raf kinase activity was the difference in the activities in tubes A and B, as P100-associated Raf-1 and B-Raf are insoluble in 17 Nonidet P-40 (19Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Total P100 MEK activity was the difference in activities in tubes B and C. MEK-specific activity was estimated by dividing the total P100 MEK kinase activity by the amount of P100 phospho-MEK present (determined by quantitative Western analysis as described above). To measure cytosolic Raf activity, volumes of the S100 fraction proportional to the P100 aliquots assayed above were diluted into 1 ml (final volume) of buffer B (50 mm Tris-HCl (pH 7.5), 75 mm NaCl, 5 mm MgCl2, 25 mm NaF, 5 mm EGTA, 100 ॖmNaVO4, 17 Nonidet P-40, 1 mm dithiothreitol, 5 ॖg/ml aprotinin, 5 ॖg/ml leupeptin, and 5 ॖg/ml pepstatin A). Samples were rotated for 3 h at 4 °C either with 2 ॖg of anti-Raf-1 antibody plus 10 ॖl of protein G beads or with 2 ॖg of anti-B-Raf antibody plus 10 ॖl of protein A beads. The beads were washed three times with buffer B and three times with buffer A and then resuspended in kinase buffer for assaying. Kinase assays were performed on the washed beads using the coupled MEK/ERK assay as described above. To ensure comparable results, P100 and S100 fractions were assayed at the same time using the same reaction buffers and then quantified simultaneously by phosphorimaging. As the P100 and S100 fractions analyzed were equivalent, total measurable Raf kinase activity was determined by adding total P100, S100 Raf-1, and S100 B-Raf kinase activities together. This value is referred to as total Raf kinase activity. Cyclin B1 kinase assays were performed as described (17Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). For immunostaining, cells were grown on poly-l-lysine-coated coverslips. Coverslips were washed with PBS, and then the cells fixed with 37 paraformaldehyde or ice-cold methanol and stored at −20 °C until required. Coverslips were washed three times with PBS and then blocked in blocking buffer (PBS containing 0.17 Tween 20 and 37 bovine serum albumin) for 1 h at room temperature. The cells were stained with anti-α-tubulin antibody (1:1000 dilution in blocking buffer), washed five times for 5 min, stained with Texas Red-conjugated anti-mouse antibody (1:300 dilution in blocking buffer containing 10 ॖg/ml 4,6-diamidino-2-phenylindole for DNA counterstaining) for 30 min, washed five times with PBS, and mounted on slides. Photomicroscopy was performed as described previously (17Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar). To examine the expression and activity of the components of the Ras/Raf/MEK/ERK pathway during cell cycle progression, we synchronized HeLa cell cultures with a double thymidine block. Using this protocol, >807 of the cells progressed synchronously through the S, G2, and M phases and into the subsequent G1 phase. By 6 h after release from the thymidine block, >857 of the cells progressed to G2 phase and, after 9 h, entered mitosis (Fig.1A). Entry and exit from mitosis were determined by measuring cyclin B1-Cdc2 kinase activity. Cyclin B1-Cdc2 activity increased at 8 h after release from the thymidine block, peaked at 9–10 h, and returned to basal levels by 12 h (Fig. 1B). These synchronized cells were used for a detailed biochemical analysis of the expression levels and activities of the endogenous components of the Ras/Raf/MEK/ERK pathway throughout the cell cycle. In HeLa cells, K-Ras and N-Ras were strongly expressed at constant levels throughout the cell cycle (Fig. 1G). H-Ras was also expressed, but at very low levels (data not shown). Ras-GTP loading was measured in a sensitive GST-Raf-RBD binding assay that permits GTP loading of each Ras isoform to be assessed simultaneously (18Clyde-Smith J. Silins G. Gartside M. Grimmond S. Etheridge M. Apolloni A. Hayward N. Hancock J.F. J. Biol. Chem. 2000; 275: 32260-32267Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). N-Ras displayed two peaks of GTP loading. The first, smaller peak occurred 12 h after release from the thymidine block and correlated with exit from mitosis. The major peak of N-Ras-GTP loading occurred in late G1phase (18 h), consistent with a role in G1/S transition (Fig. 1, C and G). In contrast, K-Ras-GTP levels remained uniformly low throughout the cell cycle, and H-Ras-GTP loading was undetectable (data not shown). Membrane-associated and cytosolic pools of Raf, MEK, and ERK have different specific activities (19Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and possibly access to different substrates. We therefore measured the activities of Raf, MEK, and ERK in membrane (P100) and cytosolic (S100) fractions. The activities of B-Raf and Raf-1 were measured in a coupled MEK/ERK kinase assay (19Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Total P100-associated Raf kinase activity and S100-associated B-Raf and Raf-1 kinase activities were assayed. The P100 Raf kinase activity profile had two peaks (Fig. 1, D and H). The first peak occurred 8–9 h after release from the thymidine block in late G2/early mitosis. Interestingly, this early peak of Raf kinase activity did not correlate with the small peak of Ras-GTP loading that occurred in late mitosis (Fig. 1, C andD). The second peak of P100 Raf kinase activity occurred in G1 phase (14–18 h) and correlated with the major peak of N-Ras-GTP loading (Fig. 1, C and D). Cytosolic B-Raf activity essentially mirrored P100-associated Raf activity, whereas cytosolic Raf-1 was not activated during mitosis, but was activated in G1 (Fig. 1H). The levels of P100 Raf-1 and B-Raf proteins were constant throughout S and G2(up to 8 h), fell during mitosis (at 9–12 h), and then increased again during G1 (at 15–18 h). The levels of cytosolic Raf-1 and B-Raf were constant (Fig. 1H). MEK activation was assayed with phospho-specific antisera that recognize activated MEK1 and MEK2 (pMEK). Two peaks of pMEK were observed. The first peak occurred in late G2/early mitosis and was entirely confined to the P100 membrane fraction (Fig. 1,E and I). The second, smaller peak occurred in G1 phase (14–18 h), but was entirely confined to the S100 fraction. Both peaks of MEK activation corresponded with peaks of Raf activity (Fig. 1, D and E). The levels of membrane-associated MEK1 and, to a lesser extent, MEK2 decreased during mitosis (10–12 h), but cytosolic MEK levels remained constant (Fig.1I). The activation of ERK was assayed in the same fractions using activation- and phospho-specific antisera (pERK). The level of pERK dramatically decreased in both P100 and S100 fractions during mitosis, but rapidly recovered during G1 progression (Fig. 1, F and J). The levels of ERK1 and ERK2 did not change throughout the cell cycle (Fig. 1J). Immunofluorescent staining for pMEK revealed strong staining of mitotic cells (Fig. 2). This mitotic pMEK staining pattern was observed in both asynchronously growing and synchronized cultures, thus was not an artifact of the synchrony protocol. The staining was dispersed throughout the cells, although weak accumulation at the spindle poles was observed in some cells (data not shown), as reported previously (11Shapiro P.S. Vaisberg E. Hunt A.J. Tolwinski N.S. Whalen A.M. McIntosh J.R. Ahn N.G. J. Cell Biol. 1998; 142: 1533-1545Crossref PubMed Scopus (194) Google Scholar). Interphase cells had low levels of phospho-MEK staining. Therefore, these results support the results from pMEK immunoblot analysis (Fig. 1, E andI). The very high level of membrane-bound pMEK in mitotic cells surprisingly correlated with a loss of active ERK, whereas by contrast, ERK was strongly activated in G1 in response to the more modest peak of MEK activity (Fig. 1, E andF). This observation strongly suggested that the coupling between MEK and ERK activation was lost in mitosis. To establish whether this loss of coupling was a direct consequence of entry into mitosis, synchronized HeLa cells were arrested in late G2phase by treatment with Compound 5, an inhibitor of the Cdc2 activator Cdc25 (20Tamura K. Southwick E.C. Kerns J. Rosi K. Carr B.I. Wilcox C. Lazo J.S. Cancer Res. 2000; 60: 1317-1325PubMed Google Scholar), or with roscovitine, a direct inhibitor of Cdc2 (21Meijer L. Borgne A. Mulner O. Chong J.P. Blow J.J. Inagaki N. Inagaki M. Delcros J.G. Moulinoux J.P. Eur. J. Biochem. 1997; 243: 527-536Crossref PubMed Scopus (1209) Google Scholar). Membrane fractions of these G2-arrested cells were compared with untreated mitotic shake-off HeLa populations by immunoblotting for pMEK and pERK using ERK2 as a protein input control. Both Cdc2 inhibitor-treated samples had reduced pMEK levels relative to normal mitotic cells, but pERK levels in the drug-treated cells were substantially higher than those in normal mitotic cells, which had barely detectable levels of pERK (Fig.3A). This result suggests that a mitotic factor either blocks access of activated MEK to ERK or directly modifies activated MEK so that it is unable to activate ERK. To differentiate between these possibilities, the activity of membrane-associated MEK was assayed in an in vitro coupled ERK1 kinase assay. Membrane-associated pMEK from roscovitine-treated G2-arrested cells had a 10-fold higher specific activity than pMEK from control mitotic cell populations (Fig. 3B). These results strongly suggest that activated MEK is directly modified as cells enter mitosis to inhibit its ability to activate ERK. Earlier studies that examined MEK activity in mitosis (10Hayne C. Tzivion G. Luo Z. J. Biol. Chem. 2000; 275: 31876-31882Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Colanzi A. Deerinck T.J. Ellisman M.H. Malhotra V. J. Cell Biol. 2000; 149: 331-339Crossref PubMed Scopus (88) Google Scholar) did not detect the dramatic MEK activation shown in Fig.1I, but these studies analyzed only detergent lysates of mitotic cells. We therefore tested the detergent solubility of P100-associated pMEK. P100 fractions prepared from mitotic HeLa cells were solubilized in 17 Nonidet P-40, and the soluble and insoluble fractions were immunoblotted for pMEK, MEK1, and MEK2. pMEK was insoluble in 17 Nonidet P-40, whereas MEK1 was highly soluble, and MEK2 had low solubility (Fig.4A). To determine which MEK isoform was activated during mitosis, the Nonidet P-40-insoluble P100 fraction of mitotic HeLa cells was electrophoresed on high resolving SDS-polyacrylamide gel and then immunoblotted for pMEK with antibodies raised against the N and C termini of MEK1 (anti-MEK1N and anti-MEK1C antibodies) and MEK2 (anti-MEK2N and anti-MEK2C antibodies). The phosphorylated form of MEK migrated with an apparent molecular mass of ∼37 kDa. Anti-MEK1C antibody detected three bands, a 44-kDa band corresponding to unmodified MEK1 and cytosolic pMEK and two more rapidly migrating bands, the most abundant of which comigrated with the 37-kDa band detected by anti-pMEK antibody (Fig. 4B). Anti-MEK1N, anti-MEK2N, and anti-MEK2C antibodies did not detect a 37-kDa band. To confirm that the 37-kDa band detected by anti-pMEK antibody was indeed MEK1, the mitotic P100 fraction was resolved by two-dimensional isoelectric focusing/SDS-PAGE and then immunoblotted for pMEK and for MEK1 and MEK2 with an antibody that detects both MEK isoforms (anti-MEK1/2 antibody). pMEK exactly comigrated with the minor species detected by anti-MEK1/2 antibody, confirming the identity of the species as a truncated form of pMEK1 (Fig. 4C). To determine the timing of MEK1 cleavage, the Nonidet P-40-insoluble P100 fraction from the experiment shown in Fig. 1I was run on high resolving SDS-polyacrylamide gel and immunoblotted with anti-MEK1C antibody. Full-length 44-kDa MEK1 was the major species in S and G2, but 37-kDa MEK1 increased in late G2 to become the major form in mitosis (Fig.5A). As cells exited mitosis and entered G1, the shorter form was lost, and the full-length 44-kDa form of MEK1 reappeared. This suggested that MEK1 was cleaved during mitosis and that the cleaved protein was stable only during mitosis. To confirm that MEK1 cleavage was a mitosis-specific event, synchronized HeLa cells were allowed to progress into mitosis or were arrested in G2 by addition of the Cdc2 inhibitor roscovitine. P100 fractions from these cells were then immunoblotted with anti-pMEK, anti-MEK1C, and anti-MEK1N antibodies. Blocking cyclin B-Cdc2 activity and entry into mitosis reduced MEK activation and MEK1 cleavage (Fig. 5B), confirming that MEK1 cleavage is a mitosis-specific event that is dependent on cyclin B-Cdc2 kinase activity. The detection of mitotic pMEK1 with only anti-MEK1C and anti-MEK1/2 antibodies suggested that the faster migrating species was the result of proteolytic cleavage of the N-terminal region of MEK1. The apparent size difference between full-length 44-kDa MEK1 and mitotic pMEK1 was 5–7 kDa, indicating that the cleavage occurred in the region of residues 40–60. This was investigated using a MEK1 internal deletion mutant with residues 32–51 removed (MEK1Δ32–51). HeLa cells were transfected with HA-tagged versions of wild-type MEK1 and MEK1Δ32–51 and either untreated or arrested in mitosis with nocodazole, which produced essentially identical accumulation of the faster migrating pMEK species observed in normal mitotic samples. S100 and P100 fractions were prepared and immunoblotted for the HA-tagged proteins. The level of HA-tagged wild-type MEK1 in the P100 fraction from mitotic cells was reduced compared with the untreated asynchronously growing culture (Fig.6A). This paralleled the reduction in MEK1 detected with anti-MEK1N antibody in normal mitotic P100 fractions (Fig. 5B). There was little change in the level of HA-tagged MEK1Δ32–51 in the P100 fractions from asynchronous and nocodazole-arrested mitotic cells (Fig.6A). The levels of these proteins were constant in the S100 fractions from asynchronous and mitotic cells. The insensitivity of the MEK1Δ32–51 constructs to N-terminal cleavage suggested either that the deleted residues 32–51 contain the proteolytic cleavage site or that the tertiary structure of the deleted protein masks the cleavage site. To discriminate between these possibilities, the N-terminal 51 residues of MEK1 were replaced with either an HA tag (HA-MEK1ΔN51) or GFP (GFP-MEK1ΔN51). Immunoblotting of the P100 fraction prepared from mitotic cells expressing GFP-MEK1ΔN51 revealed the presence of the full-length chimera, detected by both anti-MEK1C and anti-GFP antibodies, and an abundant 37-kDa MEK1 protein, detected only by anti-MEK1C antibody and corresponding to MEK1ΔN51 cle" @default.
• W2020921289 created "2016-06-24" @default.
• W2020921289 creator A5037758372 @default.
• W2020921289 creator A5038559109 @default.
• W2020921289 creator A5043907665 @default.
• W2020921289 creator A5059604768 @default.
• W2020921289 creator A5077272790 @default.
• W2020921289 date "2003-05-01" @default.
• W2020921289 modified "2023-10-18" @default.
• W2020921289 title "Mechanism of Mitosis-specific Activation of MEK1" @default.
• W2020921289 cites W1553801822 @default.
• W2020921289 cites W1925896202 @default.
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