New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

APOPTOSIS ◽  
2007 ◽  
Vol 12 (10) ◽  
pp. 1847-1855 ◽  
Author(s):  
Karen Higginbottom ◽  
Ulrike Jahnke ◽  
Adrian C. Newland ◽  
Finbarr E. Cotter ◽  
Paul David Allen
Keyword(s):  
Cyclin A ◽  
Human Cells ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Induced Apoptosis

scholarly journals Preventing the Degradation of Mps1 at Centrosomes Is Sufficient to Cause Centrosome Reduplication in Human Cells

2007 ◽  
Vol 18 (11) ◽  
pp. 4457-4469 ◽  
Author(s):  
Christopher Kasbek ◽  
Ching-Hui Yang ◽  
Adlina Mohd Yusof ◽  
Heather M. Chapman ◽  
Mark Winey ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Proteasome Inhibition ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Centrosome Duplication ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Cdk2 Activity ◽  
Necessary And Sufficient

Supernumerary centrosomes promote the assembly of abnormal mitotic spindles in many human tumors. In human cells, overexpression of the cyclin-dependent kinase (Cdk)2 partner cyclin A during a prolonged S phase produces extra centrosomes, called centrosome reduplication. Cdk2 activity protects the Mps1 protein kinase from proteasome-mediated degradation, and we demonstrate here that Mps1 mediates cyclin A-dependent centrosome reduplication. Overexpression of cyclin A or a brief proteasome inhibition increases the centrosomal levels of Mps1, whereas depletion of Cdk2 leads to the proteasome-dependent loss of Mps1 from centrosomes only. When a Cdk2 phosphorylation site within Mps1 (T468) is mutated to alanine, Mps1 cannot accumulate at centrosomes or participate in centrosome duplication. In contrast, phosphomimetic mutations at T468 or deletion of the region surrounding T468 prevent the proteasome-dependent removal of Mps1 from centrosomes in the absence of Cdk2 activity. Moreover, cyclin A-dependent centrosome reduplication requires Mps1, and these stabilizing Mps1 mutations cause centrosome reduplication, bypassing cyclin A. Together, our data demonstrate that the region surrounding T468 contains a motif that regulates the accumulation of Mps1 at centrosomes. We suggest that phosphorylation of T468 attenuates the degradation of Mps1 at centrosomes and that preventing this degradation is necessary and sufficient to cause centrosome reduplication in human cells.

scholarly journals Rescue of a Human Cell Line from Endogenous Cdk1 Depletion by Cdk1 Lacking Inhibitory Phosphorylation Sites

2006 ◽  
Vol 282 (7) ◽  
pp. 4301-4309 ◽  
Author(s):  
Mita Gupta ◽  
Deborah Trott ◽  
Andrew C. G. Porter
Keyword(s):  
Cell Line ◽  
Human Cell ◽  
Kinase Activity ◽  
Threshold Model ◽  
Human Cell Line ◽  
S Phase ◽  
Human Cells ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase

Cells that transiently overexpress cyclin-dependent kinase 1 lacking inhibitory phosphorylation sites (Cdk1-AF) undergo premature and catastrophic mitosis, reflecting the key role for Cdk1 in promoting a timely transit from G2 into mitosis. Conversely, cells depleted of Cdk1 undergo repeated S phases without intervening mitoses (endoreduplication), reflecting a role for Cdk1 in preventing premature S phases. It is not known how Cdk1 prevents entry into S phase at times in G2 when it does not promote mitosis. Also uncertain is the extent of redundancy between inhibitory phosphorylation and other mechanisms for controlling Cdk1 activity. We describe here human cells that not only tolerate stable Cdk1-AF expression but also rely on it for survival when endogenous Cdk1 is depleted. When residual endogenous Cdk1 expression is further depleted, however, proliferation of Cdk1-AF-rescued cells is inhibited. Interestingly, this inhibition is not accompanied by endoreduplication. These results are consistent with a two-threshold model for Cdk1 kinase activity, one for suppressing endoreduplication and one for promoting mitosis. They also indicate that inhibitory phosphorylation is indispensable for only a fraction of the total cellular complement of Cdk1.

THE DYNACTOME: INVESTIGATING DYNAMIC KINASE NETWORKS IN CELLULAR DECISION-MAKING

2008 ◽  
Vol 31 (4) ◽  
pp. 22
Author(s):  
Jonathan So ◽  
Kelly Elder ◽  
Anna Dai ◽  
Claus Jorgensen ◽  
Rune Linding ◽  
...  
Keyword(s):  
Spectrometric Analysis ◽  
Phosphorylation Sites ◽  
Complex Samples ◽  
Downstream Signaling ◽  
Through Flow ◽  
Colorectal Cancer Cells ◽  
Induced Apoptosis ◽  
Cell Phenotypes ◽  
Kinase Phosphorylation ◽  
Kinase Expression

Networks of kinases play a role in the transmission and integration of signals from the membrane to the nucleus. We aim to elucidate kinase phosphorylation and interaction partners in these networks through the immuno-precipitation and mass spectrometric analysis of a representative set of 100 Flag-tagged kinases stably expressed in human colorectal cancer cells. The goal is to generate a comprehensive set of interactions and dynamic phosphorylation sites which correlate with cell phenotypes such as apoptosis and proliferation. The techniques of mass-spectrometry have allowed for the identification of proteins and their phosphorylation sites in complex samples. Various labeling methods such as iTRAQ has enabled the relative quantification of these sites as afunction of time (White et al. PNAS, 2007). However, kinases usually work in the context of particular signaling stimuli. We aim to characterize the role of these over-expressed kinases in the context of Trail-induced apoptosis. This isparticularly relevant to tumorigenesis in that many cancers are resistant to apoptosis and recombinant Trail therapies are currently undergoing clinical trials. We present assays to correlate the proliferative ability and sensitivity to apoptosis of various stable cell lines with kinase expression levels through flow cytometry. We also present efforts to trace downstream signaling through the monitoring of MAP kinase phosphorylation using a high-throughput bead array.

Truncation and Optimisation of Peptide Inhibitors of Cyclin-Dependent Kinase 2-Cyclin A Through Structure-Guided Design

ChemMedChem ◽  
2009 ◽  
Vol 4 (7) ◽  
pp. 1120-1128 ◽  
Author(s):  
George Kontopidis ◽  
Martin J. Andrews ◽  
Campbell McInnes ◽  
Andy Plater ◽  
Lorraine Innes ◽  
...  
Keyword(s):  
Cyclin A ◽  
Peptide Inhibitors ◽  
Cyclin Dependent Kinase

Sex Steroids Do Not Prevent Amylin-Induced Apoptosis in Human Cells

1998 ◽  
Vol 241 (1) ◽  
pp. 265-268 ◽  
Author(s):  
A. Schwingshackl ◽  
I. Blasko ◽  
E. Steiner ◽  
P. Pozzilli ◽  
M.G. Cavallo ◽  
...  
Keyword(s):  
Sex Steroids ◽  
Human Cells ◽  
Induced Apoptosis

scholarly journals Rhotekin 2 silencing inhibits proliferation and induces apoptosis in human osteosarcoma cells

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Xiong Wang ◽  
Lei Zhang ◽  
Wenji Wang ◽  
Yuchen Wang ◽  
Ye Chen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Phase Cell ◽  
Osteosarcoma Cell ◽  
Cyclin Dependent Kinase ◽  
Potential Therapeutic Target ◽  
Human Osteosarcoma ◽  
Cycle Arrest ◽  
Human Osteosarcoma Cells ◽  
Human Osteosarcoma Cell ◽  
Induced Apoptosis

Human osteosarcoma is the most frequent primary malignant of bone, and often occurs in adolescents. However, molecular mechanism of this disease remains unclear. In the present study, we found that the level of Rhotekin 2 (RTKN2) was up-regulated in osteosarcoma tissues and cell lines. In addition, silencing of RTKN2 of human osteosarcoma cell lines U2OS, inhibited proliferation, and induced G1 phase cell cycle arrest via reducing the level of the cyclin-dependent kinase 2 (CDK2). Furthermore, RTKN2 knockdown in the U2OS cells induced apoptosis by increasing the level of Bax and decreasing the level of Bcl2. These results suggested that RTKN2 is involved in the progression of human osteosarcoma, and may be a potential therapeutic target.

scholarly journals Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells

2021 ◽  
Author(s):  
Mike T. Veling ◽  
Dan T. Nguyen ◽  
Nicole N. Thadani ◽  
Michela E. Oster ◽  
Nathan J. Rollins ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Human Cells ◽  
Disordered Proteins ◽  
Cellular Protection ◽  
Intrinsically Disordered ◽  
Apoe Protein ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Induced Apoptosis

ABSTRACTMany organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally-occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically-induced apoptosis. We show that proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlight the ability for DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and human cellular protection.

scholarly journals p27kip1 (Cyclin-Dependent Kinase Inhibitor 1B) Controls Ovarian Development by Suppressing Follicle Endowment and Activation and Promoting Follicle Atresia in Mice

2007 ◽  
Vol 21 (9) ◽  
pp. 2189-2202 ◽  
Author(s):  
Singareddy Rajareddy ◽  
Pradeep Reddy ◽  
Chun Du ◽  
Lian Liu ◽  
Krishna Jagarlamudi ◽  
...  
Keyword(s):  
Premature Ovarian Failure ◽  
Molecular Mechanisms ◽  
Ovarian Development ◽  
Ovarian Follicle ◽  
Cyclin A ◽  
Ovarian Failure ◽  
Cyclin Dependent Kinase ◽  
Female Reproduction ◽  
Oocyte Growth ◽  
Follicle Atresia

Abstract In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27kip1 or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27−/−) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27+/+ mice. Moreover, in p27−/− ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27−/− ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27−/− ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.

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