Sil Phosphorylation in a Pin1 Binding Domain Affects the Duration of the Spindle Checkpoint

ABSTRACT SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.

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Cell cycle-dependent repetitive Ca(2+)waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization

Journal of Cell Science ◽

10.1242/jcs.113.19.3453 ◽

2000 ◽

Vol 113 (19) ◽

pp. 3453-3462 ◽

Cited By ~ 4

Author(s):

A. McDougall ◽

M. Levasseur ◽

A.J. O'Sullivan ◽

K.T. Jones

Keyword(s):

Spatial Pattern ◽

Kinase Activity ◽

Cyclin B1 ◽

Egg Activation ◽

Second Phase ◽

Protein Factor ◽

Sperm Extract ◽

Two Phases ◽

Cycle Dependent ◽

Cytosolic Factor

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+)waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

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The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

The Journal of Cell Biology ◽

10.1083/jcb.200912084 ◽

2010 ◽

Vol 190 (4) ◽

pp. 587-602 ◽

Cited By ~ 32

Author(s):

Wouter van Zon ◽

Janneke Ogink ◽

Bas ter Riet ◽

René H. Medema ◽

Hein te Riele ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Spindle Checkpoint ◽

Cyclin A ◽

Cyclin B1 ◽

Mitotic Exit ◽

Dependent Manner ◽

Anaphase Promoting Complex ◽

Checkpoint Control ◽

N Terminus ◽

Mitotic Phosphorylation

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1–Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/CCdc20 makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.

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Mechanisms of G2 Arrest in Response to Overexpression of p53

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3607 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3607-3622 ◽

Cited By ~ 110

Author(s):

William R. Taylor ◽

Samuel E. DePrimo ◽

Archana Agarwal ◽

Munna L. Agarwal ◽

Axel H. Schönthal ◽

...

Keyword(s):

Constitutive Expression ◽

Cyclin B1 ◽

Cyclin Dependent Kinase ◽

G2 Arrest ◽

Cdc2 Activity ◽

In Cells ◽

Constitutively Active

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 andcyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions −287 to −123 of thecyclin B1 promoter and −104 to −74 of thecdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest.

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Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

Biology of Reproduction ◽

10.1093/biolre/ioab019 ◽

2021 ◽

Author(s):

Cecilia Valencia ◽

Felipe Alonso Pérez ◽

Carola Matus ◽

Ricardo Felmer ◽

María Elena Arias

Keyword(s):

Protein Synthesis ◽

Kinase Activity ◽

Cyclin B1 ◽

Protein Synthesis Inhibitors ◽

Vitro Fertilization ◽

New Findings ◽

Bovine Oocytes ◽

Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

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Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells

Communications Biology ◽

10.1038/s42003-021-02035-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Ilaria Frasson ◽

Paola Soldà ◽

Matteo Nadai ◽

Sara Lago ◽

Sara N. Richter

Keyword(s):

Transcription Factor ◽

Early Gene ◽

Proximity Ligation Assay ◽

Gene Promoters ◽

Herpes Simplex Virus 1 ◽

Direct Binding ◽

Simplex Virus ◽

In Cells ◽

Hsv 1

AbstractG-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

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Protein tyrosine phosphatase-α complexes with the IGF-I receptor and undergoes IGF-I-stimulated tyrosine phosphorylation that mediates cell migration

AJP Cell Physiology ◽

10.1152/ajpcell.00110.2009 ◽

2009 ◽

Vol 297 (1) ◽

pp. C133-C139 ◽

Cited By ~ 11

Author(s):

Shirley C. Chen ◽

Ranvikram S. Khanna ◽

Darrell C. Bessette ◽

Lionel A. Samayawardhena ◽

Catherine J. Pallen

Keyword(s):

Cell Migration ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Kinases ◽

Tyrosine Phosphatase ◽

Phosphorylation Site ◽

Protein Tyrosine ◽

Fibroblast Migration ◽

Igf I ◽

In Cells

Protein tyrosine phosphatase-α (PTPα) is a widely expressed receptor-type phosphatase that functions in multiple signaling systems. The actions of PTPα can be regulated by its phosphorylation on serine and tyrosine residues, although little is known about the conditions that promote PTPα phosphorylation. In this study, we tested the ability of several extracellular factors to stimulate PTPα tyrosine phosphorylation. The growth factors IGF-I and acidic FGF induced the highest increase in PTPα phosphorylation at tyrosine 789, followed by PMA and lysophosphatidic acid, while EGF had little effect. Further investigation of IGF-I-induced PTPα tyrosine phosphorylation demonstrated that this occurs through a novel Src family kinase-independent mechanism that does not require focal adhesion kinase, phosphatidylinositol 3-kinase, or MEK. We also show that PTPα physically interacts with the IGF-I receptor. In contrast to IGF-I-induced PTPα phosphorylation, this association does not require IGF-I. The interaction of PTPα and the IGF-I receptor is independent of PTPα catalytic activity, and expression of exogenous PTPα does not promote IGF-I receptor tyrosine dephosphorylation, indicating that PTPα does not act as an IGF-I receptor phosphatase. However, PTPα mediates IGF-I signaling, because IGF-I-stimulated fibroblast migration was reduced by ∼50% in cells lacking PTPα or in cells with mutant PTPα lacking the tyrosine 789 phosphorylation site. Our results suggest that PTPα tyrosine phosphorylation can occur in response to diverse stimuli and can be mediated by various tyrosine kinases. In the case of IGF-I, we propose that IGF-I-induced tyrosine 789 phosphorylation of PTPα, possibly catalyzed by the PTPα-associated IGF-I receptor tyrosine kinase, is required for efficient cell migration in response to this growth factor.

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Increased cyclin B1/CDC 2 kinase activity and phosphorylation of Bcl-2 associated with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells

APOPTOSIS ◽

10.1007/bf01321020 ◽

1996 ◽

Vol 1 (2) ◽

pp. 141-146 ◽

Cited By ~ 4

Author(s):

C-H. Shu ◽

W. K. Yang ◽

T-S. Huang

Keyword(s):

Nasopharyngeal Carcinoma ◽

Kinase Activity ◽

Cyclin B1 ◽

Carcinoma Cells ◽

Human Nasopharyngeal Carcinoma ◽

Induced Apoptosis ◽

Cdc 2

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Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47729-0 ◽

1987 ◽

Vol 262 (32) ◽

pp. 15341-15344 ◽

Cited By ~ 15

Author(s):

J Hari ◽

R A Roth

Keyword(s):

Kinase Activity ◽

Insulin Receptors ◽

In Cells

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Activation of c-Src in cells bearing v-Crk and its suppression by Csk

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4706-4713.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4706-4713

Author(s):

H Sabe ◽

M Okada ◽

H Nakagawa ◽

H Hanafusa

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Src Kinase ◽

Cellular Protein ◽

Morphological Transformation ◽

Protein Tyrosine ◽

In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

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