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 Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway

2008 ◽  
Vol 28 (17) ◽  
pp. 5172-5183 ◽  
Author(s):  
Tetsuro Horie ◽  
Kazuo Tatebayashi ◽  
Rika Yamada ◽  
Haruo Saito
Keyword(s):  
Protein Kinase ◽  
Active Form ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Function ◽  
Response Regulators ◽  
High Osmolarity ◽  
Inactive Form ◽  
Mitogen Activated Protein ◽  
Mutant Phenotypes

ABSTRACT In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1∼P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1∼P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.

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 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 Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2 Phase and Promotes the G2/M Cell Cycle Transition

2007 ◽  
Vol 18 (2) ◽  
pp. 594-604 ◽  
Author(s):  
Timothy N. Feinstein ◽  
Adam D. Linstedt
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Cell Cycle Checkpoint ◽  
Mitogen Activated Protein Kinase ◽  
M Cell ◽  
M Phase ◽  
Mitogen Activated Protein ◽  
Cell Cycle Transition ◽  
Protein Kinase Kinase

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G2/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle “checkpoint.” A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G2 to control G2/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G2 Golgi unlinking and the G2/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.

scholarly journals Epithelial Cell Cycle Behaviour in the Injured Kidney

2018 ◽  
Vol 19 (7) ◽  
pp. 2038 ◽  
Author(s):  
Lies Moonen ◽  
Patrick D’Haese ◽  
Benjamin Vervaet
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Mitogen Activated Protein Kinase ◽  
Cell Cycle Regulators ◽  
M Phase ◽  
Mitogen Activated Protein ◽  
Proximal Tubular

Acute kidney injury (AKI), commonly caused by ischemia-reperfusion injury, has far-reaching health consequences. Despite the significant regenerative capacity of proximal tubular epithelium cells (PTCs), repair frequently fails, leading to the development of chronic kidney disease (CKD). In the last decade, it has been repeatedly demonstrated that dysregulation of the cell cycle can cause injured kidneys to progress to CKD. More precisely, severe AKI causes PTCs to arrest in the G1/S or G2/M phase of the cell cycle, leading to maladaptive repair and a fibrotic outcome. The mechanisms causing these arrests are far from known. The arrest might, at least partially, be attributed to DNA damage since activation of the DNA-damage response pathway leads to cell cycle arrest. Alternatively, cytokine signalling via nuclear factor kappa beta (NF-κβ) and p38-mitogen-activated protein kinase (p38-MAPK) pathways, and reactive oxygen species (ROS) can play a role independent of DNA damage. In addition, only a handful of cell cycle regulators (e.g., p53, p21) have been thoroughly studied during renal repair. Still, why and how PTCs decide to arrest their cell cycle and how this arrest can efficiently be overcome remain open and challenging questions. In this review we will discuss the evidence for cell cycle involvement during AKI and development of CKD together with putative therapeutic approaches.

scholarly journals Stage-specific Requirement of a Mitogen-activated Protein Kinase by Trypanosoma brucei

2002 ◽  
Vol 13 (11) ◽  
pp. 3787-3799 ◽  
Author(s):  
Ingrid B. Müller ◽  
Debora Domenicali-Pfister ◽  
Isabel Roditi ◽  
Erik Vassella
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Tsetse Fly ◽  
Mitogen Activated Protein Kinase ◽  
Mammalian Host ◽  
Null Mutant ◽  
African Trypanosomes ◽  
Mitogen Activated Protein

In cycling between the mammalian host and the tsetse fly vector, African trypanosomes undergo adaptive differentiation steps that are coupled to growth control. The signaling pathways underlying these cellular processes are largely unknown. Mitogen-activated protein kinases (MAPKs) are known mediators of growth and differentiation in other eukaryotic organisms. To establish the function of a MAPK homologue, TbMAPK2, in T. brucei, a null mutant was constructed. Bloodstream forms of aΔmapk2/Δmapk2 clone were able to grow normally and exhibited no detectable phenotype. When these cells were triggered to differentiate in vitro, however, they developed to the procyclic (fly midgut) form with delayed kinetics and subsequently underwent cell cycle arrest. Introduction of an ectopic copy of theTbMAPK2 gene into the null mutant restored its ability to differentiate and to divide. In contrast, a TbMAPK2mutant, in which the T190 and Y192 residues of the activating phosphorylation site were replaced by A and F, was unable to restore the growth and differentiation phenotypes. Analysis of the DNA content and the nucleus/kinetoplast configuration of individual cells showed that the null mutant was arrested in all phases of the cell cycle and that 25–30% of the cells had failed to segregate their nucleus and kinetoplast correctly. This implies that cell cycle progression by the procyclic form depends on a constitutive stimulus exerted by the signaling cascade operating through TbMAPK2.

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.

Sign in / Sign up

Export Citation Format

Share Document