scholarly journals Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.

1993 ◽  
Vol 4 (2) ◽  
pp. 195-208 ◽  
Author(s):  
H J Yoon ◽  
S Loo ◽  
J L Campbell
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Protein A ◽  
Yeast Cells ◽  
Protein Kinase Activity ◽  
Protein Levels ◽  
Phosphorylation Pattern ◽  
Phosphopeptide Mapping

The yeast Cdc7 function is required for the G1/S transition and is dependent on passage through START, a point controlled by the Cdc28/cdc2/p34 protein kinase. CDC7 encodes a protein kinase activity, and we now show that this kinase activity varies in the cell cycle but that protein levels appear to remain constant. We present several lines of evidence that periodic activation of CDC7 kinase is at least in part through phosphorylation. First, the kinase activity of the Cdc7 protein is destroyed by dephosphorylation of the protein in vitro with phosphatase. Second, Cdc7 protein is hypophosphorylated and inactive as a kinase in extracts of cells arrested at START but becomes active and maximally phosphorylated subsequent to passage through START. The phosphorylation pattern of Cdc7 protein is complex. Phosphopeptide mapping reveals four phosphopeptides in Cdc7 prepared from asynchronous yeast cells. Both autophosphorylation and phosphorylation in trans appear to contribute to this pattern. Autophosphorylation is shown to occur by using a thermolabile Cdc7 protein. A protein in yeast extracts can phosphorylate and activate Cdc7 protein made in Escherichia coli, and phosphorylation is thermolabile in cdc28 mutant extracts. Cdc7 protein carrying a serine to alanine change in the consensus recognition site for Cdc28 kinase shows an altered phosphopeptide map, suggesting that this site is important in determining the overall Cdc7 phosphorylation pattern.

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.

scholarly journals Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

1991 ◽  
Vol 115 (5) ◽  
pp. 1275-1282 ◽  
Author(s):  
T Usui ◽  
M Yoshida ◽  
K Abe ◽  
H Osada ◽  
K Isono ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
S Phase ◽  
Continuous Treatment ◽  
Protein Kinase Activity

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

scholarly journals The Cak1p Protein Kinase Is Required at G1/S and G2/M in the Budding Yeast Cell Cycle

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 57-71 ◽  
Author(s):  
Ann Sutton ◽  
Richard Freiman
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Synthetic Lethal ◽  
Protein Levels ◽  
M Phase

Abstract The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G2 to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G2 to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G1 into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function.

scholarly journals A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4.

1994 ◽  
Vol 14 (5) ◽  
pp. 3350-3363 ◽  
Author(s):  
J Weinstein ◽  
F W Jacobsen ◽  
J Hsu-Chen ◽  
T Wu ◽  
L G Baum
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Yeast Cells ◽  
Protein Kinase Activity ◽  
Growing Cell ◽  
Dividing Cells

A novel protein, p55CDC, has been identified in cycling mammalian cells. This transcript is readily detectable in all exponentially growing cell lines but disappears when cells are chemically induced to fall out of the cell cycle and differentiate. The p55CDC protein appears to be essential for cell division, since transfection of antisense p55CDC cDNA into CHO cells resulted in isolation of only those cells which exhibited a compensatory increase in p55CDC transcripts in the sense orientation. Immunoprecipitation of p55CDC yielded protein complexes with kinase activity which fluctuated during the cell cycle. Since p55CDC does not have the conserved protein kinase domains, this activity must be due to one or more of the associated proteins in the immune complex. The highest levels of protein kinase activity were seen with alpha-casein and myelin basic protein as substrates and demonstrated a pattern of activity distinct from that described for the known cyclin-dependent cell division kinases. The p55CDC protein was also phosphorylated in dividing cells. The amino acid sequence of p55CDC contains seven repeats homologous to the beta subunit of G proteins, and the highest degree of homology in these repeats was found with the Saccharomyces cerevisiae Cdc20 and Cdc4 proteins, which have been proposed to be involved in the formation of a functional bipolar mitotic spindle in yeast cells. The G beta repeat has been postulated to mediate protein-protein interactions and, in p55CDC, may modulate its association with a unique cell cycle protein kinase. These findings suggest that p55CDC is a component of the mammalian cell cycle mechanism.

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.

A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4

1994 ◽  
Vol 14 (5) ◽  
pp. 3350-3363
Author(s):  
J Weinstein ◽  
F W Jacobsen ◽  
J Hsu-Chen ◽  
T Wu ◽  
L G Baum
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Yeast Cells ◽  
Protein Kinase Activity ◽  
Growing Cell ◽  
Dividing Cells

A novel protein, p55CDC, has been identified in cycling mammalian cells. This transcript is readily detectable in all exponentially growing cell lines but disappears when cells are chemically induced to fall out of the cell cycle and differentiate. The p55CDC protein appears to be essential for cell division, since transfection of antisense p55CDC cDNA into CHO cells resulted in isolation of only those cells which exhibited a compensatory increase in p55CDC transcripts in the sense orientation. Immunoprecipitation of p55CDC yielded protein complexes with kinase activity which fluctuated during the cell cycle. Since p55CDC does not have the conserved protein kinase domains, this activity must be due to one or more of the associated proteins in the immune complex. The highest levels of protein kinase activity were seen with alpha-casein and myelin basic protein as substrates and demonstrated a pattern of activity distinct from that described for the known cyclin-dependent cell division kinases. The p55CDC protein was also phosphorylated in dividing cells. The amino acid sequence of p55CDC contains seven repeats homologous to the beta subunit of G proteins, and the highest degree of homology in these repeats was found with the Saccharomyces cerevisiae Cdc20 and Cdc4 proteins, which have been proposed to be involved in the formation of a functional bipolar mitotic spindle in yeast cells. The G beta repeat has been postulated to mediate protein-protein interactions and, in p55CDC, may modulate its association with a unique cell cycle protein kinase. These findings suggest that p55CDC is a component of the mammalian cell cycle mechanism.

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells

1985 ◽  
Vol 5 (10) ◽  
pp. 2647-2652
Author(s):  
C A Cartwright ◽  
M A Hutchinson ◽  
W Eckhart
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Tumor Antigen ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Amino Terminal ◽  
Exogenous Substrate ◽  
And Function ◽  
Amino Terminal Domain

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.

scholarly journals Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

1985 ◽  
Vol 5 (10) ◽  
pp. 2543-2551 ◽  
Author(s):  
I MacDonald ◽  
J Levy ◽  
T Pawson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Peripheral Blood ◽  
Hematopoietic Cells ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Sarcoma Virus ◽  
Human And Mouse ◽  
Specific Protein Kinase

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

Sign in / Sign up

Export Citation Format

Share Document