scholarly journals Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

2015 ◽  
Vol 26 (16) ◽  
pp. 2913-2926 ◽  
Author(s):  
Daniel P. Miller ◽  
Hana Hall ◽  
Ryan Chaparian ◽  
Madison Mara ◽  
Alison Mueller ◽  
...  
Keyword(s):  
Ring Formation ◽  
Cyclin Dependent Kinase ◽  
Filamentous Actin ◽  
Actin Ring ◽  
Ring Assembly ◽  
Cdk Phosphorylation ◽  
Necessary And Sufficient ◽  
Type Ii Myosin ◽  
Actin Rings

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.

scholarly journals Estrogen Decreases Cytoskeletal Organization by Forming an ERα/SHP2/c-Src Complex in Osteoclasts to Protect against Ovariectomy-Induced Bone Loss in Mice

Antioxidants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 619
Author(s):  
Hyun-Jung Park ◽  
Malihatosadat Gholam-Zadeh ◽  
Sun-Young Yoon ◽  
Jae-Hee Suh ◽  
Hye-Seon Choi
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Ring Formation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Actin Ring ◽  
Collagen Crosslinks ◽  
Trap Staining ◽  
Src Activation

Loss of ovarian function is closely related to estrogen (E2) deficiency, which is responsible for increased osteoclast (OC) differentiation and activity. We aimed to investigate the action mechanism of E2 to decrease bone resorption in OCs to protect from ovariectomy (OVX)-induced bone loss in mice. In vivo, tartrate-resistant acid phosphatase (TRAP) staining in femur and serum carboxy-terminal collagen crosslinks-1 (CTX-1) were analyzed upon E2 injection after OVX in mice. In vitro, OCs were analyzed by TRAP staining, actin ring formation, carboxymethylation, determination of reactive oxygen species (ROS) level, and immunoprecipitation coupled with Western blot. In vivo and in vitro, E2 decreased OC size more dramatically than OC number and Methyl-piperidino-pyrazole hydrate dihydrochloride (MPPD), an estrogen receptor alpha (ERα) antagonist, augmented the OC size. ERα was found in plasma membranes and E2/ERα signaling affected receptor activator of nuclear factor κB ligand (RANKL)-induced actin ring formation by rapidly decreasing a proto-oncogene tyrosine-protein kinase, cellular sarcoma (c-Src) (Y416) phosphorylation in OCs. E2 exposure decreased physical interactions between NADPH oxidase 1 (NOX1) and the oxidized form of c-Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), leading to higher levels of reduced SHP2. ERα formed a complex with the reduced form of SHP2 and c-Src to decrease c-Src activation upon E2 exposure, which blocked a signal for actin ring formation by decreased Vav guanine nucleotide exchange factor 3 (Vav3) (p–Y) and Ras-related C3 botulinum toxin substrate 1 (Rac1) (GTP) activation in OCs. E2/ERα signals consistently inhibited bone resorption in vitro. In conclusion, our study suggests that E2-binding to ERα forms a complex with SHP2/c-Src to attenuate c-Src activation that was induced upon RANKL stimulation in a non-genomic manner, resulting in an impaired actin ring formation and reducing bone resorption.

scholarly journals TNFα Potently Activates Osteoclasts, through a Direct Action Independent of and Strongly Synergistic with RANKL

Endocrinology ◽  
2002 ◽  
Vol 143 (3) ◽  
pp. 1108-1118 ◽  
Author(s):  
Karen Fuller ◽  
Chiho Murphy ◽  
Barrie Kirstein ◽  
Simon W. Fox ◽  
Timothy J. Chambers
Keyword(s):  
Direct Action ◽  
Ring Formation ◽  
Actin Ring ◽  
Independent Manner ◽  
Low Concentrations ◽  
Order Of Magnitude ◽  
Extreme Sensitivity ◽  
Osteoclastic Differentiation

Abstract TNFα is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFα promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFα promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats. TNFα was equipotent with RANKL for this action. Activation by TNFα was unaffected by blockade of RANKL by OPG, its soluble decoy receptor, suggesting that this was due to a direct action on osteoclasts. Bone resorption was similarly directly and potently stimulated, in a RANKL-independent manner in osteoclasts, whether these were formed in vitro or in vivo. Interestingly, TNFα promoted actin ring formation at concentrations an order of magnitude below those required for osteoclastic differentiation. Moreover, TNFα strongly synergized with RANKL, such that miniscule concentrations of TNFα were sufficient to substantially augment osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNFα suggests that the most sensitive osteolytic response of bone to TNFα is through activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism whereby increased osteolysis can be achieved without disturbance to the underlying pattern of osteoclastic localization.

Actin rings in cytokinesis of apical cells in red algae

1996 ◽  
Vol 74 (6) ◽  
pp. 971-974 ◽  
Author(s):  
David J. Garbary ◽  
A. Rosa McDonald
Keyword(s):  
Cell Division ◽  
Red Algae ◽  
Ring Formation ◽  
Transverse Wall ◽  
Actin Ring ◽  
Dense Ring ◽  
Red Algal ◽  
First Time ◽  
Actin Rings ◽  
Peripheral Cytoplasm

Actin rings are demonstrated for the first time in florideophyte red algae in Audouinella botryocarpa (Acrochaetiales) and Tiffaniella snyderae and Griffithsia pacifica (Ceramiales). Rings formed a complete circle in the peripheral cytoplasm prior to the development of the septum by furrowing. In the uninucleate A. botryocarpa, ring formation precedes mitosis. In all species F-actin initially has a diffuse to longitudinal alignment throughout the cell. The actin realigns and then contracts to form a dense actin ring. Following cell division, aggregations of actin are maintained along the transverse wall in at least G. pacifica and A. botryocarpa, and a dense ring of actin was observed around a pit plug in T. snyderae. The occurrence of actin rings in cell division of members of the Acrochaetiales and Ceramiales is consistent with ultrastructural observations on red algal unicells and suggests that actin rings are part of the ancestral cytokinetic mechanism in red algae. Keywords: actin rings, cell division, cytokinesis, cytoskeleton, microfilaments, Rhodophyta.

scholarly journals Of Bars and Rings: Hof1-Dependent Cytokinesis in Multiseptated Hyphae of Ashbya gossypii

2008 ◽  
Vol 29 (3) ◽  
pp. 771-783 ◽  
Author(s):  
Andreas Kaufmann ◽  
Peter Philippsen
Keyword(s):  
Ashbya Gossypii ◽  
Actin Ring ◽  
Src Homology 3 ◽  
Filamentous Ascomycete ◽  
Multinucleated Cells ◽  
Ring Assembly ◽  
Src Homology ◽  
Type Ii Myosin ◽  
First Time

ABSTRACT We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.

scholarly journals Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

2001 ◽  
Vol 12 (12) ◽  
pp. 4044-4053 ◽  
Author(s):  
Daniel P. Mulvihill ◽  
Caroline Barretto ◽  
Jeremy S. Hyams
Keyword(s):  
Fission Yeast ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Ring Formation ◽  
Restrictive Temperature ◽  
Actin Ring ◽  
Septation Initiation Network ◽  
Green Fluorescent ◽  
Type Ii Myosin ◽  
The Relationship

Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S1518A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S1518E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25°C but not at 36°C. GFP-Myo2S1518E rings persisted at 36°C incdc7-24 but not in another SIN kinase mutant,sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy ofmyo2+was fused to GFP (strainmyo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36°C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.

Elevation of fibrinogen due to loss of ovarian function enhances actin ring formation and leads to increased bone resorption

2012 ◽  
Vol 303 (11) ◽  
pp. E1296-E1303 ◽  
Author(s):  
Ke Ke ◽  
Woon-Ki Kim ◽  
Ok-Joo Sul ◽  
Van Tien Phan ◽  
Mi-Hyun Lee ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Ovarian Function ◽  
Annexin V ◽  
Ring Formation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Actin Ring ◽  
Macrophage Colony ◽  
And Function

The aim of the present study was to evaluate the effect of fibrinogen on number and function of osteoclasts (OC) consequently resulting in bone loss. It was hypothesized that the enhanced level of released fibrinogen due to loss of ovarian function caused bone loss by acting on OCs. Bone loss was induced by ovariectomy (OVX) in mice and analyzed by micro-CT. The effect of fibrinogen on OCs was evaluated by tartrate-resistant acid phosphatase, annexin V, actin staining, pit formation observed on dentine slices, and Western blotting. Exogenous fibrinogen increased OC survival, actin ring formation, and bone resorption in vitro. The effect of fibrinogen was dependent on β3-integrin, which is a marker for mature OCs. Fibrinogen induced the activation of transforming oncogene from Ak strain (Akt), Ras-related C3 botulinum toxin substrate 1 (Rac1), and Rho family of GTPase (Rho) and the degradation of the Bcl-2 interacting mediator of cell death (Bim) in a manner similar to macrophage colony-stimulating factor (M-CSF). OVX increased plasma fibrinogen and serum M-CSF together with elevated actin ring formation and bone loss. The increased fibrinogen level due to loss of ovarian function may contribute, at least partly, to bone loss through the enhanced number and activity of OCs.

scholarly journals Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on 3 sites increases transactivation potency and stimulates cell proliferation

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1193-1200 ◽  
Author(s):  
Linsheng Zhang ◽  
Florence B. Fried ◽  
Hong Guo ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Assay ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Inhibition Of Proliferation ◽  
Cdk Phosphorylation ◽  
Kinase Phosphorylation ◽  
Lineage Specific Genes

Abstract RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and stimulates G1 cell-cycle progression. Within RUNX1, S48, S303, and S424 fit the cyclin-dependent kinase (cdk) phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cdks on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now use an in vitro kinase assay, phosphopeptide-specific antiserum, and the cdk inhibitor roscovitine to demonstrate that S48 and S424 are also phosphorylated by cdk1 or cdk6 in hematopoietic cells. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell-cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation of the cdk sites to the partially phosphomimetic aspartic acid mildly reduced DNA affinity while progressively increasing transactivation of a model reporter. Mutation to alanine increased DNA affinity, suggesting that in other gene or cellular contexts phosphorylation of RUNX1 by cdks may reduce transactivation. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Together these findings indicate that cdk phosphorylation of RUNX1 potentially couples stem/progenitor proliferation and lineage progression.

scholarly journals Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation

2006 ◽  
Vol 399 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Chantelle Sedgwick ◽  
Matthew Rawluk ◽  
James Decesare ◽  
Sheetal Raithatha ◽  
James Wohlschlegel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phase Boundary ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Proliferating Cells ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb–Cdc28 inhibitor Sic1. In proliferating cells Cln–Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCFCdc4 and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCFCdc4 was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCFCdc4. In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

scholarly journals Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

1998 ◽  
Vol 9 (9) ◽  
pp. 2393-2405 ◽  
Author(s):  
Masafumi Nishizawa ◽  
Masaoki Kawasumi ◽  
Marie Fujino ◽  
Akio Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Cdk Inhibitor ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cdk Phosphorylation ◽  
Cdc28 Kinase

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

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