14-3-3zeta cooperates with Phosphorylated Plk1 and is required for correct cytokinesis
Proteins of the 14-3-3 family are functionally conserved in eukaryotic kingdom which participates in diversified and critical cellular processes. However, the exact roles of these proteins in mitotic regulation has remained elusive. Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that plays multiple critical functions such as centrosome maturation, mitotic chromosome segregation, cytokinesis, and the DNA damage response. Here we show that 14-3-3zeta interacts and cooperates with Plk1 in mitotic progress. 14-3-3zeta is associated with the spindle at metaphase and concentrated in the midbody during cytokinesis. Using yeast two hybrid assay, we found a functional connection between 14-3-3zeta and Plk1. We demonstrate that phosphorylation of Plk1 at S330 and S597 promotes its interaction with 14-3-3zeta. Importantly, 14-3-3zeta cooperates with Plk1 in ensuring successful cytokinesis. We conclude that mitotic phosphorylation of Plk1 promotes interaction with 14-3-3zeta and this interaction is required for faithful cytokinesis. Taken together with the results of previous studies, our results suggest 14-3-3 family emerges as a novel player in mitotic regulation: cooperation with Plk1 to ensure a faithful cytokinesis.
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ABSTRACT: Objective. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate the activities of target mRNAs and cellular processes. miR-451 is one of miRNAs conserved perfectly among vertebrates and regulates cell proliferation, invasion, and apoptosis in tumor. However, the role of miR-451 in autoimmune arthritis has been unknown. Our study was designed to identify the role of miR-451 in autoimmune arthritis. Methods. We compared the expression of miR-451 in neutrophils from patients with rheumatoid arthritis (RA) and healthy controls (HCs). The role of miR-451 in neutrophil chemotaxis was evaluated in vivo and in vitro using neutrophils of mice. The regulation of p38 mitogen-activated protein kinase by miR-451 was assessed. Arthritis score and histology in SKG mice were examined by the administration of double-stranded miR-451. Results. miR-451 expression was lower in neutrophils isolated from patients with RA than in those from HCs. Systemic administration of miR-451 significantly disturbed the infiltration of neutrophils in air pouch model without affecting apoptosis of neutrophils. Overexpression of miR-451 significantly suppressed the migration of neutrophils to formyl-methionyl-leucyl-phenylalanine. We identified CPNE3 and Rab5a as direct targets of miR-451. Overexpression of miR-451 suppressed the phosphorylation of p38 mitogen-activated protein kinase (MAPK) via 14-3-3ζ, a known target of miR-451, and Rab5a. In SKG mice, miR-451 treatment reduced the severity of arthritis and the number of infiltrating cells. Conclusions. These results suggest that miR-451 suppresses neutrophil chemotaxis via p38 MAPK and that miR-451 is a potential therapeutic target in the treatment of RA. © 2013 American College of Rheumatology.
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ABSTRACT: The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remains unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12 and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.
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The 14-3-3 (YWHA) proteins are highly conserved in higher eukaryotes, participate in various cellular signaling pathways including cell cycle regulation, development and growth. Our previous studies demonstrated that 14-3-3¿ (YWHAE) is responsible for maintaining prophase | arrest in mouse oocyte. However, roles of 14-3-3¿ in the mitosis of fertilized mouse eggs have remained unclear. Here, we showed that 14-3-3¿ interacts and cooperates with CDC25B phosphorylated at Ser321 regulating G2/M transition of mitotic progress of fertilized mouse eggs.ResultsDisruption of 14-3-3¿ expression by RNAi prevented normal G2/M transition by inhibition of MPF activity and leaded to the translocation of CDC25B into the nucleus from the cytoplasm. Overexpression of 14-3-3¿-WT and unphosphorylatable CDC25B mutant (CDC25B-S321A) induced mitotic resumption in dbcAMP-arrested eggs. In addition, we examined endogenous and exogenous distribution of 14-3-3¿ and CDC25B. Endogenous 14-3-3¿ and CDC25B were co-localized primarily in the cytoplasm at the G1, S, early G2 and M phases whereas CDC25B was found to accumulate in the nucleus at the late G2 phase. Upon coexpression with RFP¿14-3-3¿, GFP¿CDC25B¿WT and GFP¿CDC25B¿S321A were predominantly cytoplasmic at early G2 phase and then GFP¿CDC25B¿S321A moved to the nucleus whereas CDC25B-WT signals were observed in the cytoplasm without nucleus accumulation at late G2 phase at presence of dbcAMP.Conclusions
Our data indicate that 14-3-3¿ is required for the mitotic entry in the fertilized mouse eggs. 14-3-3¿ is primarily responsible for sequestering the CDC25B in cytoplasm and 14-3-3¿ binding to CDC25B-S321 phosphorylated by PKA induces mitotic arrest at one-cell stage by inactivation of MPF in fertilized mouse eggs.
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