scholarly journals The SIT4 protein phosphatase functions in late G1 for progression into S phase.

1991 ◽  
Vol 11 (4) ◽  
pp. 2133-2148 ◽  
Author(s):  
A Sutton ◽  
D Immanuel ◽  
K T Arndt
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Function Analysis ◽  
S Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Amino Terminal ◽  
Polymorphic Gene ◽  
Cycle Dependent

Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.

The SIT4 protein phosphatase functions in late G1 for progression into S phase

1991 ◽  
Vol 11 (4) ◽  
pp. 2133-2148
Author(s):  
A Sutton ◽  
D Immanuel ◽  
K T Arndt
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Function Analysis ◽  
S Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Amino Terminal ◽  
Polymorphic Gene ◽  
Cycle Dependent

Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.

scholarly journals The Human Papillomavirus Type 18 E2 Protein Is a Cell Cycle-Dependent Target of the SCFSkp2 Ubiquitin Ligase

2009 ◽  
Vol 84 (1) ◽  
pp. 437-444 ◽  
Author(s):  
Sophie Bellanger ◽  
Chye Ling Tan ◽  
Wenlong Nei ◽  
Ping Ping He ◽  
Françoise Thierry
Keyword(s):  
Cell Cycle ◽  
Human Papillomavirus ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Major Step ◽  
Direct Role ◽  
Amino Terminal ◽  
E6 And E7 ◽  
Cycle Dependent ◽  
Phase Entry

ABSTRACT The human papillomavirus type 18 (HPV-18) E2 gene is inactivated in cervical carcinoma after integration of the viral DNA into the host cellular genome. Since E2 represses the transcription of the two viral oncogenes E6 and E7, integration which allows their strong expression is considered a major step in transformation by HPV. We show here that E2 is specifically degraded at the end of the G1 phase in a Brd4-independent manner, implying that its regulatory functions are cell cycle dependent. Degradation of E2 occurs via the Skp1/Cullin1/F-box Skp2 (SCFSkp2) ubiquitin ligase, since silencing of Skp2 induces stabilization of E2. In addition, the amino-terminal domain of E2 can interact with Skp2 as shown by coimmunoprecipitation experiments. We previously showed that E2 inhibits the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, leading to accumulation of several of its substrates. We demonstrate here that Skp2, which is a known APC/C substrate in G1, is also stabilized by E2. Therefore, by negative feedback, SCFSkp2 activation could lead to E2 degradation and E6/E7 expression specifically in the late G1 phase. Moreover, since the SCFSkp2 can trigger S-phase entry and Skp2 itself is a known oncogene, we believe that E2-mediated accumulation of Skp2, together with E2 degradation leading to putative release of E6 and E7 inhibition, could induce premature S-phase entry in HPV-infected cells, pointing to a direct role of E2 in the early steps of HPV-mediated transformation.

TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells

1997 ◽  
Vol 110 (2) ◽  
pp. 179-189 ◽  
Author(s):  
T. Asada ◽  
R. Kuriyama ◽  
H. Shibaoka
Keyword(s):  
Cell Cycle ◽  
Preprophase Band ◽  
S Phase ◽  
Motor Domain ◽  
Bipolar Structure ◽  
Amino Terminal ◽  
M Phase ◽  
Microtubule Motor ◽  
Cycle Dependent ◽  
In Cells

Analysis of a cDNA for a 125 kDa polypeptide, previously isolated from phragmoplasts of tobacco BY-2 cells as a candidate for a plus end-directed microtubule motor, revealed this polypeptide to be a novel member of the kinesin superfamily. We named this protein TKRP125 (tobacco kinesin-related polypeptide of 125 kDa). The strong similarity between TKRP125 and members of the bimC subfamily in terms of the amino acid sequence of the amino-terminal motor domain indicated that TKRP125 belonged to the bimC subfamily. An antibody against a short peptide from the motor domain of TKRP125 inhibited the GTP- or ATP-dependent translocation of phragmoplast microtubules in membrane-permeabilized BY-2 cells, suggesting a role for TKRP125 in microtubule translocation, which is considered to be involved in the formation and/or maintenance of the bipolar structure of the phragmoplast. The expression of TKRP125 was found to be cell cycle-dependent. TKRP125 was not present in cells at the G1 phase. It began to appear at the S phase and accumulated during the G2 phase. The distribution of TKRP125 changed as the arrangement of microtubules changed with the progression of the cell cycle. TKRP125 was distributed along cortical microtubules during the S phase and along microtubules in the preprophase band and perinuclear microtubules in premitotic cells. It was also present in the nucleus in premitotic cells. In cells in M phase, TKRP125 was distributed along spindle microtubules. It accumulated at the equatorial plane of the spindle as the spindle elongated. In cytokinetic cells, TKRP125 was colocalized with phragmoplast microtubules. These observations suggest the possible involvement of TKRP125 in the cell cycle-dependent changes in arrays of microtubules, including the organization of the phragmoplast, and in the movement of chromosomes in anaphase cells.

scholarly journals A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle.

1995 ◽  
Vol 128 (6) ◽  
pp. 1131-1144 ◽  
Author(s):  
E Sontag ◽  
V Nunbhakdi-Craig ◽  
G S Bloom ◽  
M C Mumby
Keyword(s):  
Cell Cycle ◽  
Enzymatic Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
S Phase ◽  
Phosphatase 2A ◽  
Spindle Microtubules ◽  
Cycle Dependent ◽  
Cycle Regulation

Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport.

scholarly journals Cell cycle–dependent localization of macroH2A in chromatin of the inactive X chromosome

2002 ◽  
Vol 157 (7) ◽  
pp. 1113-1123 ◽  
Author(s):  
Brian P. Chadwick ◽  
Huntington F. Willard
Keyword(s):  
Cell Cycle ◽  
X Chromosome ◽  
Degradation Pathway ◽  
S Phase ◽  
Histone H2a ◽  
The Core ◽  
Inactive X Chromosome ◽  
Inactivation Center ◽  
Chromatin Proteins ◽  
Cycle Dependent

One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

scholarly journals Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

2012 ◽  
Vol 23 (21) ◽  
pp. 4203-4211 ◽  
Author(s):  
Dong-Hwan Kim ◽  
Deanna M. Koepp
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Charged Residues ◽  
Phase Progression ◽  
Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

scholarly journals Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells

1985 ◽  
Vol 225 (2) ◽  
pp. 529-533 ◽  
Author(s):  
A J Strain ◽  
W A H Wallace ◽  
A H Wyllie
Keyword(s):  
Cell Cycle ◽  
Gene Transfer ◽  
Transfection Efficiency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
S Phase ◽  
Sv40 Virus ◽  
G2 Phase ◽  
Cycle Dependent ◽  
Sv40 Dna

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.

scholarly journals The kinetoplastid-specific phosphatase KPP1 attenuates PLK activity to facilitate flagellum inheritance in Trypanosoma brucei

2021 ◽  
Vol 14 (669) ◽  
pp. eabc6435
Author(s):  
Tai An ◽  
Huiqing Hu ◽  
Ziyin Li
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Complex Structure ◽  
Cell Communication ◽  
Specific Protein ◽  
Activation Loop ◽  
Human Parasite ◽  
Complex Protein

Trypanosoma brucei, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled flagellum during the cell cycle requires the Polo-like kinase homolog TbPLK and the kinetoplastid-specific protein phosphatase KPP1, although whether TbPLK acts on KPP1 or vice versa has been unclear. Here, we showed that dephosphorylation of TbPLK on Thr125 by KPP1 maintained low TbPLK activity in the flagellum-associated hook complex structure, thereby ensuring proper flagellum positioning and attachment. This dephosphorylation event required the recognition of phosphorylated Thr198 in the activation loop of TbPLK by the N-terminal Plus3 domain of KPP1 and the dephosphorylation of phosphorylated Thr125 in TbPLK by the C-terminal catalytic domain of KPP1. Dephosphorylation of TbPLK by KPP1 prevented hyperphosphorylation of the hook complex protein TbCentrin2, thereby allowing timely dephosphorylation of phosphorylated TbCentrin2 for hook complex duplication and flagellum positioning and attachment. Thus, KPP1 attenuates TbPLK activity by dephosphorylating TbPLK to facilitate flagellum inheritance.

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