scholarly journals Inhibitor-2 induced M-phase arrest in Xenopus cycling egg extracts is dependent on MAPK activation

2011 ◽  
Vol 16 (4) ◽  
Author(s):  
Arian Khandani ◽  
Mahmood Mohtashami ◽  
Anne Camirand
Keyword(s):  
Cell Cycle ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Protein Phosphatase 1 ◽  
Mapk Activity ◽  
Phase Arrest ◽  
Evolutionarily Conserved ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein

AbstractThe evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle.

scholarly journals Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in CyclingXenopus Egg Extracts

1998 ◽  
Vol 9 (2) ◽  
pp. 451-467 ◽  
Author(s):  
John C. Bitangcol ◽  
Andrew S.-S. Chau ◽  
Ellamae Stadnick ◽  
Manfred J. Lohka ◽  
Bryan Dicken ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Mapk Signaling ◽  
Cyclin B ◽  
Mitogen Activated Protein Kinase ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein ◽  
Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.

scholarly journals Confluence of Vascular Endothelial Cells Induces Cell Cycle Exit by Inhibiting p42/p44 Mitogen-Activated Protein Kinase Activity

1999 ◽  
Vol 19 (4) ◽  
pp. 2763-2772 ◽  
Author(s):  
Francesc Viñals ◽  
Jacques Pouysségur
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Vascular Endothelial Cells ◽  
Mitogen Activated Protein Kinase ◽  
Vascular Endothelial ◽  
Cell Cycle Exit ◽  
Mapk Activation ◽  
Mapk Activity ◽  
Mitogen Activated Protein

ABSTRACT Like other cellular models, endothelial cells in cultures stop growing when they reach confluence, even in the presence of growth factors. In this work, we have studied the effect of cellular contact on the activation of p42/p44 mitogen-activated protein kinase (MAPK) by growth factors in mouse vascular endothelial cells. p42/p44 MAPK activation by fetal calf serum or fibroblast growth factor was restrained in confluent cells in comparison with the activity found in sparse cells. Consequently, the induction of c-fos, MAPK phosphatases 1 and 2 (MKP1/2), and cyclin D1 was also restrained in confluent cells. In contrast, the activation of Ras and MEK-1, two upstream activators of the p42/p44 MAPK cascade, was not impaired when cells attained confluence. Sodium orthovanadate, but not okadaic acid, restored p42/p44 MAPK activity in confluent cells. Moreover, lysates from confluent 1G11 cells more effectively inactivated a dually phosphorylated active p42 MAPK than lysates from sparse cells. These results, together with the fact that vanadate-sensitive phosphatase activity was higher in confluent cells, suggest that phosphatases play a role in the down-regulation of p42/p44 MAPK activity. Enforced long-term activation of p42/p44 MAPK by expression of the chimera ΔRaf-1:ER, which activates the p42/p44 MAPK cascade at the level of Raf, enhanced the expression of MKP1/2 and cyclin D1 and, more importantly, restored the reentry of confluent cells into the cell cycle. Therefore, inhibition of p42/p44 MAPK activation by cell-cell contact is a critical step initiating cell cycle exit in vascular endothelial cells.

333 DYNAMICS OF MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITY IN PIG OOCYTES FOLLOWING PARTHENOGENETIC ACTIVATION BY DIFFERENT METHODS

2007 ◽  
Vol 19 (1) ◽  
pp. 282
Author(s):  
L. Nanassy ◽  
K. Lee ◽  
A. Javor ◽  
Z. Machaty
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Pronuclear Formation

Cell cycle progression during mitosis and meiosis is known to be regulated by the M-phase promoting factor (MPF). However, recent findings revealed that mitogen-activated protein kinase (MAPK) also plays an important regulatory role during transition through the cell cycle. At fertilization the activity of MAPK drops shortly after MPF inactivation; the objective of this study was to investigate the dynamics of MAPK activity in pig oocytes after different activation methods. In vitro-matured oocytes were allocated to 3 groups. In group 1 (EP), the oocytes were activated by 2 DC pulses of 1.2 kV cm-1, 60 �s each. In the second group (EP + BU), the oocytes were electroporated and incubated for 4 h in 100 �M butyrolactone I (BU, an inhibitor of cdc2 kinase). In group 3 (EP + CHX), the oocytes were electroporated and treated for 5 h with 10 �g mL-1 cycloheximide (CHX, a protein synthesis inhibitor). After electroporation all oocytes were incubated in 7.5 �g mL-1 cytochalasin B for 4 h. Some oocytes were used to determine MAPK activity at 0, 1, 2, 3, 4, 5, and 6 h after electroporation using a MAPK assay kit. The assay measures MAPK activity by determining the phosphorylation of myelin basic protein by MAPK using the transfer of the γ-phosphate of [γ-32P] ATP. Pronuclear formation was evaluated at 6 h after electroporation; blastocyst formation and total cell numbers per embryo were determined after a 7-day culture in PZM-3 medium. Pronuclear formation was compared by the chi-square test, blastocyst formation was assessed using ANOVA, and the kinase activity was evaluated using the Student t-test. Pronuclear formation was highest in the combined methods [69.39% (EP) vs. 86.32% (EP + BU) and 87.56 % (EP + CHX); P < 0.05]. Similarly, the combined methods supported better development to the blastocyst stage [25.06 � 7.96% (EP), 58.32 � 7.62% (EP + BU), and 63.91 � 6.35% (EP + CHX); P < 0.05], whereas the average cell numbers of the blastocysts did not differ (47.11 � 3.12, 46.56 � 2.33, and 44.04 � 1.86, respectively). The initial MAPK activity was 0.123 � 0.017 pmol/min/oocyte which, after 1 h, dropped in all cases to values of 0.069 � 0.009 (EP), 0.072 � 0.007 (EP + BU), and 0.077 � 0.012 (EP + CHX) pmol/min/oocyte (P < 0.05). The MAPK activity in the EP group reached its lowest level at 3 h (0.057 � 0.007 pmol/min/oocyte); however, at 4 h it started to recover and by 6 h the activity (0.079 � 0.022 pmol/min/oocyte) did not differ from that of the non-activated oocytes. In the other groups, MAPK activity stayed low, and by the end of the experimental period it was significantly lower than that in the nontreated metaphase II oocytes (P < 0.05). The results indicate that electroporation followed by protein kinase inhibition or protein synthesis inhibition leads to the efficient inactivation of MAPK activity, and confirm our earlier findings that these combined treatments support superior embryo development after oocyte activation.

scholarly journals Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly

2008 ◽  
Vol 181 (2) ◽  
pp. 241-254 ◽  
Author(s):  
Michael J. Emanuele ◽  
Weijie Lan ◽  
Miri Jwa ◽  
Stephanie A. Miller ◽  
Clarence S.M. Chan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Culture Cells ◽  
Kinetochore Assembly ◽  
M Phase ◽  
Egg Extracts ◽  
Cycle Dependent

The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle–dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

scholarly journals Transcriptional Coregulation by the Cell Integrity Mitogen-Activated Protein Kinase Slt2 and the Cell Cycle Regulator Swi4

2001 ◽  
Vol 21 (19) ◽  
pp. 6515-6528 ◽  
Author(s):  
Kristin Baetz ◽  
Jason Moffat ◽  
Jennifer Haynes ◽  
Michael Chang ◽  
Brenda Andrews
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Wall ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Subunit ◽  
Cell Integrity ◽  
Mitogen Activated Protein

ABSTRACT In Saccharomyces cerevisiae, the heterodimeric transcription factor SBF (for SCB binding factor) is composed of Swi4 and Swi6 and activates gene expression at the G1/S-phase transition of the mitotic cell cycle. Cell cycle commitment is associated not only with major alterations in gene expression but also with highly polarized cell growth; the mitogen-activated protein kinase (MAPK) Slt2 is required to maintain cell wall integrity during periods of polarized growth and cell wall stress. We describe experiments aimed at defining the regulatory pathway involving the cell cycle transcription factor SBF and Slt2-MAPK. Gene expression assays and chromatin immunoprecipitation experiments revealed Slt2-dependent recruitment of SBF to the promoters of the G1 cyclinsPCL1 and PCL2 after activation of the Slt2-MAPK pathway. We performed DNA microarray analysis and identified other genes whose expression was reduced in both SLT2and SWI4 deletion strains. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated and reveal a role for Swi4, the DNA-binding component of SBF, which is independent of the regulatory subunit Swi6. Some of the Swi4- and Slt2-dependent genes do not require Swi6 for either their expression or for Swi4 localization to their promoters. Consistent with these results, we found a direct interaction between Swi4 and Slt2. Our results establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond its prominent role in controlling cell cycle transcription.

scholarly journals Roles of Greatwall Kinase in the Regulation of Cdc25 Phosphatase

2008 ◽  
Vol 19 (4) ◽  
pp. 1317-1327 ◽  
Author(s):  
Yong Zhao ◽  
Olivier Haccard ◽  
Ruoning Wang ◽  
Jiangtao Yu ◽  
Jian Kuang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Xenopus Oocytes ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Entry ◽  
M Phase ◽  
Egg Extracts ◽  
Mitogen Activated Protein ◽  
Xenopus Egg ◽  
Greatwall Kinase ◽  
Xenopus Egg Extracts

We previously reported that immunodepletion of Greatwall kinase prevents Xenopus egg extracts from entering or maintaining M phase due to the accumulation of inhibitory phosphorylations on Thr14 and Tyr15 of Cdc2. M phase–promoting factor (MPF) in turn activates Greatwall, implying that Greatwall participates in an MPF autoregulatory loop. We show here that activated Greatwall both accelerates the mitotic G2/M transition in cycling egg extracts and induces meiotic maturation in G2-arrested Xenopus oocytes in the absence of progesterone. Activated Greatwall can induce phosphorylations of Cdc25 in the absence of the activity of Cdc2, Plx1 (Xenopus Polo-like kinase) or mitogen-activated protein kinase, or in the presence of an activator of protein kinase A that normally blocks mitotic entry. The effects of active Greatwall mimic in many respects those associated with addition of the phosphatase inhibitor okadaic acid (OA); moreover, OA allows cycling extracts to enter M phase in the absence of Greatwall. Taken together, these findings support a model in which Greatwall negatively regulates a crucial phosphatase that inhibits Cdc25 activation and M phase induction.

scholarly journals Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies

2004 ◽  
Vol 378 (3) ◽  
pp. 857-865 ◽  
Author(s):  
Hyukjin CHA ◽  
Chad HANCOCK ◽  
Surabhi DANGI ◽  
Dony MAIGUEL ◽  
France CARRIER ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Active Form ◽  
Phosphorylation Site ◽  
Cross Reactivity ◽  
Mitogen Activated Protein Kinase ◽  
Peptide Sequence ◽  
Specific Antibodies ◽  
M Phase ◽  
Mitogen Activated Protein ◽  
35 Kda Protein

Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells. These findings reveal a new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/MKK2 antibodies to examine the regulation of MKK1/MKK2 during mitotic transitions, owing to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.

scholarly journals The mitogen-activated protein kinase pathway tonically inhibits both basal and IGF-I-stimulated IGF-binding protein-5 production in mammary epithelial cells

2007 ◽  
Vol 194 (2) ◽  
pp. 349-359 ◽  
Author(s):  
Jodie M Fleming ◽  
Jeffrey A Brandimarto ◽  
Wendie S Cohick
Keyword(s):  
Mammary Epithelial Cells ◽  
Mapk Pathway ◽  
Mammary Epithelial ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activity ◽  
Igf I ◽  
Mitogen Activated Protein ◽  
Igf Binding ◽  
Mapk Inhibitors ◽  
Igfbp 3

The IGF system plays a key role in mammary gland growth and development. Our lab previously reported that IGF-I primarily regulates IGF-binding protein (BP)-3 in bovine mammary epithelial cells (MEC) and IGFBP-5 in mammary fibroblasts (MF). Presently, we examined the signaling pathways used by IGF-I to elicit this distinct, cell-type specific regulation. The phosphatidylinositol-3 kinase pathway was required for IGF-I to increase IGFBP-3 and -5 in MF and IGFBP-3 in MEC. Surprisingly, inhibiting the mitogen-activated protein kinase (MAPK) pathway in MEC increased IGFBP-5 mRNA levels 2- to 4-fold under basal conditions and 8- to 12-fold in cells treated with IGF-I within 4 h. Similar patterns of IGFBP-3 and -5 regulation were observed in murine MEC. Cells treated with IGF-I in the presence of MAPK inhibitors secreted more IGFBP-5 protein into conditioned media relative to cells treated with IGF-I alone; however, IGFBP-5 protein was not detected in conditioned media of cells treated with only a MAPK inhibitor. The IGFBP-5 mRNA response to MAPK inhibitors was specific for MEC, as blocking MAPK activity decreased the ability of IGF-I to induce IGFBP-5 in MF. In addition, no other IGFBP was increased in either cell type when MAPK activity was inhibited. These increases in IGFBP-5 expression in response to inhibition of the MAPK pathway corresponded with the induction of apoptosis. In conclusion, we report the novel observation that the MAPK/extracellular signal regulated kinase (ERK) pathway specifically represses IGFBP-5 expression in MEC. The corresponding changes in apoptosis and IGFBP-5 expression support a role for this specific IGFBP in mammary gland involution.

scholarly journals Scaffolding Protein Gab2 Mediates Differentiation Signaling Downstream of Fms Receptor Tyrosine Kinase

2001 ◽  
Vol 21 (9) ◽  
pp. 3047-3056 ◽  
Author(s):  
Yan Liu ◽  
Brendan Jenkins ◽  
Jung Lim Shin ◽  
Larry R. Rohrschneider
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Mapk Pathway ◽  
Growth Potential ◽  
Mitogen Activated Protein Kinase ◽  
Scaffolding Protein ◽  
Macrophage Differentiation ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Macrophage Colony

ABSTRACT Fms is the receptor for macrophage colony-stimulating factor (M-CSF) and contains intrinsic tyrosine kinase activity. Expression of exogenous Fms in a murine myeloid progenitor cell line, FDC-P1 (FD-Fms), results in M-CSF-dependent growth and macrophage differentiation. Previously, we described a 100-kDa protein that was tyrosine phosphorylated upon M-CSF stimulation of FD-Fms cells. In this report, we identify this 100-kDa protein as the recently cloned scaffolding protein Gab2, and we demonstrate that Gab2 associates with several molecules involved in M-CSF signaling, including Grb2, SHP2, the p85 subunit of phosphatidylinositol 3′-kinase, SHIP, and SHC. Tyrosine phosphorylation of Gab2 in response to M-CSF requires the kinase activity of Fms, but not that of Src. Overexpression of Gab2 in FD-Fms cells enhanced both mitogen-activated protein kinase (MAPK) activity and macrophage differentiation, but reduced proliferation, in response to M-CSF. In contrast, a mutant of Gab2 that is unable to bind SHP2 did not potentiate MAPK activity. Furthermore, overexpression of this mutant in FD-Fms cells inhibited macrophage differentiation and resulted in a concomitant increase in growth potential in response to M-CSF. These data indicate that Gab2 is involved in the activation of the MAPK pathway and that the interaction between Gab2 and SHP2 is essential for the differentiation signal triggered by M-CSF.

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