scholarly journals Stress-mediated exit to quiescence restricted by increasing persistence in CDK4/6 activation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Hee Won Yang ◽  
Steven D Cappell ◽  
Ariel Jaimovich ◽  
Chad Liu ◽  
Mingyu Chung ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Single Cells ◽  
S Phase ◽  
Reporter System ◽  
Dependent Protein Kinase ◽  
Cell Cycle Entry ◽  
Cdk2 Activity ◽  
Dependent Protein ◽  
Time Periods ◽  
Stress Signals

Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here, we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly before CDK2 activity gradually increases, and that CDK4/6 activity can be active after mitosis or inactive for variable time periods. Markedly, stress signals in G1 can rapidly inactivate CDK4/6 to return cells to quiescence but with reduced probability as cells approach S phase. Together, our study reveals a regulation of G1 length by temporary inactivation of CDK4/6 activity after mitosis, and a progressively increasing persistence in CDK4/6 activity that restricts cells from returning to quiescence as cells approach S phase.

scholarly journals The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle.

1991 ◽  
Vol 11 (8) ◽  
pp. 4045-4052 ◽  
Author(s):  
S Garrett ◽  
M M Menold ◽  
J R Broach
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
S Phase ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Dependent Protein

Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase). That is, loss of the YAK1 protein specifically compensates for loss of the A kinase. Here, we show that the protein encoded by YAK1 has protein kinase activity. Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase. Induction is not observed by arrest at stages later in the cell cycle. Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity. Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity. These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs

1987 ◽  
Vol 7 (9) ◽  
pp. 3098-3106
Author(s):  
I Abraham ◽  
R J Hunter ◽  
K E Sampson ◽  
S Smith ◽  
M M Gottesman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Multiple Drug Resistance ◽  
Regulatory Subunit ◽  
Parent Cell ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Antimitotic Drug ◽  
Multiple Drugs

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

scholarly journals Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs.

1987 ◽  
Vol 7 (9) ◽  
pp. 3098-3106 ◽  
Author(s):  
I Abraham ◽  
R J Hunter ◽  
K E Sampson ◽  
S Smith ◽  
M M Gottesman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Multiple Drug Resistance ◽  
Regulatory Subunit ◽  
Parent Cell ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Antimitotic Drug ◽  
Multiple Drugs

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1–3 proteins in Saccharomyces cerevisiae

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1205-1216 ◽  
Author(s):  
Melanie Bolte ◽  
Patrick Dieckhoff ◽  
Cindy Krause ◽  
Gerhard H. Braus ◽  
Stefan Irniger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Negative Regulator ◽  
Anaphase Promoting Complex ◽  
Ras Proteins ◽  
Dependent Protein Kinase ◽  
Glucose Addition ◽  
Dependent Protein ◽  
Pka Pathway ◽  
Redundant Functions

Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis. APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases. The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins. This study shows that glucose and the activated Ras2Val19 protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast. Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways. Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium. Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function. However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3. Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway.

scholarly journals Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

1984 ◽  
Vol 4 (6) ◽  
pp. 1079-1085 ◽  
Author(s):  
J K McClung ◽  
R F Kletzien
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Cyclic Amp ◽  
Growth Kinetics ◽  
S Phase ◽  
G1 Phase ◽  
Dependent Protein Kinase ◽  
Bhk Cells ◽  
Dependent Protein ◽  
Cell Growth Kinetics

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.

scholarly journals The Anti-apoptotic Function of Hsp70 in the Interferon-inducible Double-stranded RNA-dependent Protein Kinase-mediated Death Signaling Pathway Requires the Fanconi Anemia Protein, FANCC

2002 ◽  
Vol 277 (51) ◽  
pp. 49638-49643 ◽  
Author(s):  
Qishen Pang ◽  
Tracy A. Christianson ◽  
Winifred Keeble ◽  
Tara Koretsky ◽  
Grover C. Bagby
Keyword(s):  
Protein Kinase ◽  
Fanconi Anemia ◽  
Mammalian Cells ◽  
Physical Interaction ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Multimeric Complex ◽  
Dependent Protein

Proteins encoded by five of the six known Fanconi anemia (FA) genes form a heteromeric complex that facilitates repair of DNA damage induced by cross-linking agents. A certain number of these proteins, notably FANCC, also function independently to modulate apoptotic signaling, at least in part, by suppressing ground state activation of the pro-apoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR). Because certain FANCC mutations interdict its anti-apoptotic function without interfering with the capacity of FANCC to participate functionally in the FA multimeric complex, we suspected that FANCC enhances cell survival independent of its participation in the complex. By investigating this function in both mammalian cells and in yeast, an organism with no FA orthologs, we show that FANCC inhibited the kinase activity of PKR bothin vivoandin vitro, and this effect depended upon a physical interaction between FANCC and Hsp70 but not on interactions of FANCC with other Fanconi proteins. Hsp70, FANCC, and PKR form a ternary complex in lymphoblasts and in yeast expressing PKR. We conclude that Hsp70 requires the cooperation of FANCC to suppress PKR activity and support survival of hematopoietic cells and that FANCC does not require the multimeric FA complex to exert this function.

scholarly journals Cyclic GMP-dependent Protein Kinase Activates Cloned BKCaChannels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072

1999 ◽  
Vol 274 (16) ◽  
pp. 10927-10935 ◽  
Author(s):  
Mitsuhiro Fukao ◽  
Helen S. Mason ◽  
Fiona C. Britton ◽  
James L. Kenyon ◽  
Burton Horowitz ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Mammalian Cells ◽  
Dependent Protein Kinase ◽  
Dependent Protein
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