NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1

2000 ◽  
Vol 349 (2) ◽  
pp. 509-518 ◽  
Author(s):  
Nicholas R. HELPS ◽  
Xinmei LUO ◽  
Hazel M. BARKER ◽  
Patricia T. W. COHEN
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Protein Phosphatase ◽  
Cell Cycle Progression ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Phosphatase Inhibitors ◽  
Cell Extracts ◽  
Co Precipitation

The cell cycle-regulated protein serine/threonine NIMA-related kinase 2 (Nek2), which shows a predominant localization at centrosomes, is identified as a protein which interacts with protein phosphatase 1 (PP1) using the yeast two-hybrid system. Complex formation between Nek2 and PP1 is supported by co-precipitation of the two proteins using transfected expression constructs of Nek2 and the endogenous Nek2/PP1 proteins. The sequence KVHF in the C-terminal region of Nek2, which conforms to the consensus PP1-binding motif, is shown to be essential for the interaction of Nek2 with PP1. Nek2 activity increases with autophosphorylation and addition of phosphatase inhibitors and decreases in the presence of PP1. PP1 is a substrate for Nek2 and phosphorylation of PP1γ1 on two C-terminal sites reduces its phosphatase activity. The presence of a ternary complex containing centrosomal Nek2-associated protein (C-Nap1), Nek2 and PP1 has also been demonstrated, and C-Nap1 is shown to be a substrate for both Nek2 and PP1 in vitro and in cell extracts. The implications of kinase-phosphatase complex formation involving Nek2 and PP1 are discussed in terms of the coordination of centrosome separation with cell cycle progression.

scholarly journals CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization

2019 ◽  
Vol 218 (12) ◽  
pp. 4042-4062 ◽  
Author(s):  
Reito Watanabe ◽  
Masatoshi Hara ◽  
Ei-ichi Okumura ◽  
Solène Hervé ◽  
Daniele Fachinetti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Interaction Network ◽  
Binding Motif ◽  
Cycle Progression ◽  
Kinetochore Assembly ◽  
Centromere Proteins

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A–CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A–binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A–CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

SU11657, a FLT3-Targeted Tyrosine Kinase, Has Pro-Apoptotic Activity on Leukemia Cells In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2797-2797
Author(s):  
Tiziana Grafone ◽  
Laura Ferraretti ◽  
Emanuela Ottaviani ◽  
Manuela Mancini ◽  
Michela Palmisano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Juxtamembrane Region ◽  
Cell Extracts ◽  
Relevant Effect ◽  
Wide Range

Abstract Fms-related tyrosine kinase3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has implicated in its pathogenesis. Constitutive activation of the Flt3 receptor tyrosine kinase, have been linked either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutation in the second tyrosine kinase domain (TKD). To investigate the effect in vitro of SU11657, a new compound FLT3 kinase inhibitors, we analyzed human cell lines from AML patients (MV4-11 and HL60) and blast from patients AML using a wide range of concentrations (1nM-10μM) of this novel agent. In HL-60, FLT3-wt cell line, used as negative control does not show relevant effect after treatment with SU11657. Instead, in MV4-11, FLT3-ITD cell line, we observed a decrease dose-dependent in cell viability after treatment with SU11657. The effects of this compound on cell cycle progression show an accumulation of G1/S phase and an induction of apoptosis at 1-10nM concentration after 24h of treatment. First we observed a dephosphorylation of FLT3 on Tyr591 in whole cell extracts from MV4-11 cells after treatment with SU11657 100nM. We also demonstrated a hypophosphorylation of AKT on Ser473 and a consequently dephosphorylation of BAD on Ser136 at nanomolar concentration. We observed a dephosphorylation of STAT-5 to 100nM of SU11657 at 24h. We evaluated the effects of this new compound in AML primary progenitors that showed FLT3-ITD, FLt3-TKD and FLT3-wt. In the patients with mutation ITD and TKD was evident a modification of cell cycle progression with a decrease in G2/M phase and an increase of subdiploid peak. The effect of SU11657 in patients FLT3-wt was not relevant. Our study thus showed a potential therapeutic usefulness of the drug in treatment of AML. Study of signal transductions and gene profile expression will contribute to further understanding of the drug mechanisms.

C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis

2010 ◽  
Vol 428 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Pierre-Luc Tanguay ◽  
Geneviève Rodier ◽  
Sylvain Meloche
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mitotic Cell ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Cycle Dependent

ERK3 (extracellular-signal-regulated kinase 3) is an atypical MAPK (mitogen-activated protein kinase) that is suggested to play a role in cell-cycle progression and cellular differentiation. However, it is not known whether the function of ERK3 is regulated during the cell cycle. In the present paper, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M→G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3-deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using MS analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Ser705, located at the extreme C-terminus of ERK3. All four sites are followed by a proline residue. We have shown that purified cyclin B-Cdk1 (cyclindependent kinase 1) phosphorylates these sites in vitro and demonstrate that Cdk1 acts as a major Thr698 kinase in vivo. Reciprocally, we found that the phosphatases Cdc14A and Cdc14B (Cdc is cell-division cycle) bind to ERK3 and reverse its C-terminal phosphorylation in mitosis. Importantly, alanine substitution of the four C-terminal phosphorylation sites markedly decreased the half-life of ERK3 in mitosis, thereby linking phosphorylation to the stabilization of the kinase. The results of the present study identify a novel regulatory mechanism of ERK3 that operates in a cell-cycle-dependent manner.

scholarly journals The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts

2020 ◽  
Vol 21 (16) ◽  
pp. 5709
Author(s):  
Irene Lang ◽  
Guneet Virk ◽  
Dale C. Zheng ◽  
Jason Young ◽  
Michael J. Nguyen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Phosphatase ◽  
Smooth Muscle Contraction ◽  
Cellular Systems ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Myosin Phosphatase ◽  
Phosphatase Inhibitors ◽  
Regulatory Myosin Light Chain

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

scholarly journals Protein phosphatase 1 regulatory subunit 1A regulates cell cycle progression in Ewing sarcoma

Oncotarget ◽  
2020 ◽  
Vol 11 (19) ◽  
pp. 1691-1704
Author(s):  
Wen Luo ◽  
Changxin Xu ◽  
Sarah Phillips ◽  
Aliza Gardenswartz ◽  
Jeremy M. Rosenblum ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ewing Sarcoma ◽  
Protein Phosphatase ◽  
Cell Cycle Progression ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Cycle Progression
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