The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain

1993 ◽  
Vol 105 (2) ◽  
pp. 519-528
Author(s):  
F. Boschelli ◽  
S.M. Uptain ◽  
J.J. Lightbody
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Sh2 Domain ◽  
Wild Type ◽  
Type V ◽  
Cdc28 Kinase ◽  
In Cells

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

scholarly journals Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp60v-src expressed in the yeast Saccharomyces cerevisiae.

1994 ◽  
Vol 5 (3) ◽  
pp. 283-296 ◽  
Author(s):  
M Florio ◽  
L K Wilson ◽  
J B Trager ◽  
J Thorner ◽  
G S Martin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Protein Phosphorylation ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Lethal Effect ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Inhibitory ◽  
Type V

Expression of pp60v-src, the transforming protein of Rous sarcoma virus, arrests the growth of the yeast Saccharomyces cerevisiae. To determine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes under the control of the galactose-inducible, glucose repressible GAL1 promoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expressing a catalytically inactive mutant (K295M) continued to grow normally, indicating that the kinase activity of pp60v-src is required for its growth inhibitory effect. Mutants of v-src altered in the SH2/SH3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibition of growth, indicating that complete growth inhibition requires either targeting of the active kinase or binding of the kinase to phosphorylated substrates, or both. Cells arrested by v-src expression displayed aberrant microtubule structures, alterations in DNA content and elevated p34CDC28 kinase activity. Immunoblotting with antiphosphotyrosine antibody showed that many yeast proteins, including the p34CDC28 kinase, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylation observed following v-src expression were reversed by co-expression of a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP1B). However a v-src mutant with a small insertion in the catalytic domain (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These results indicate that inappropriate phosphorylation at tyrosine is the primary cause of the lethal effect of pp60v-src expression but suggest that only a limited subset of the phosphorylated proteins are involved in this effect.

scholarly journals Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

1999 ◽  
Vol 19 (7) ◽  
pp. 4888-4896 ◽  
Author(s):  
Guy Oshiro ◽  
Julia C. Owens ◽  
Yiqun Shellman ◽  
Robert A. Sclafani ◽  
Joachim J. Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
S Phase ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Initiation Of Dna Replication

ABSTRACT In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

scholarly journals Three independent forms of regulation affect expression of HO, CLN1 and CLN2 during the cell cycle of Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 138 (4) ◽  
pp. 1015-1024 ◽  
Author(s):  
L Breeden ◽  
G Mikesell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Normal Cell ◽  
Cell Cycle Regulation ◽  
Cell Cycle Control ◽  
G1 Cyclins ◽  
Affect Expression ◽  
Cdc28 Kinase ◽  
Cycle Regulation ◽  
Normal Cell Cycle

Abstract The G1 cyclins (CLNs) bind to and activate the CDC28 kinase during the G1 to S transition in Saccharomyces cerevisiae. Two G1 cyclins are regulated at the RNA level so that their RNAs peak at the G1/S boundary. In this report we show that the cell cycle regulation of CLN1 and CLN2 is partially determined by the restricted expression of SW14, a known trans-activator of SCB elements. When SWI4 is constitutively expressed or deleted, cell cycle regulation of CLN1/2 is reduced but not eliminated. In the absence of SwI6, another known regulator of both SCB and MCB elements, cell cycle regulation of the CLNs is also reduced, and the Start-dependence of HO transcription is eliminated. This indicates that SwI6 also plays an important role in the normal cell cycle regulation of all three promoters. When both SwI6 activity and the transcriptional regulation of SW14 are eliminated, cell cycle regulation is further reduced, indicating that these are two independent pathways of regulation. However, a twofold fluctuation in transcript levels still persists under these conditions. This reveals a third source of cell cycle control, which could affect SwI4 activity post-transcriptionally, or reflect the existence of another unidentified regulator of these promoters.

Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles

Cell ◽  
1992 ◽  
Vol 70 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Yuxin Yin ◽  
Michael A. Tainsky ◽  
Farideh Z. Bischoff ◽  
Louise C. Strong ◽  
Geoffrey M. Wahl
Keyword(s):  
Cell Cycle ◽  
Gene Amplification ◽  
Cell Cycle Control ◽  
Mutant P53 ◽  
Wild Type ◽  
Wild Type P53 ◽  
In Cells

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

1994 ◽  
Vol 125 (3) ◽  
pp. 625-638 ◽  
Author(s):  
J Lukas ◽  
H Müller ◽  
J Bartkova ◽  
D Spitkovsky ◽  
A A Kjerulff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Cyclin D1 ◽  
Cell Lines ◽  
T Antigen ◽  
Regulatory Function ◽  
Retinoblastoma Gene ◽  
Checkpoint Control ◽  
Wild Type ◽  
In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

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

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.

Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5710-5717
Author(s):  
E A Malone ◽  
C D Clark ◽  
A Chiang ◽  
F Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Copy Number ◽  
Cell Cycle Control ◽  
Control Point ◽  
Temperature Sensitive ◽  
Upstream Activating Sequence ◽  
In Trans ◽  
Cis And Trans ◽  
Insertion Mutations

SPT16 was previously identified as a high-copy-number suppressor of delta insertion mutations in the 5' regions of the HIS4 and LYS2 genes of Saccharomyces cerevisiae. We have constructed null mutations in the SPT16 gene and have demonstrated that it is essential for growth. Temperature-sensitive-lethality spt16 alleles have been isolated and shown to be pleiotropic; at a temperature permissive for growth, spt16 mutations suppress delta insertion mutations, a deletion of the SUC2 upstream activating sequence, and mutations in trans-acting genes required for both SUC2 and Ty expression. In addition, SPT16 is identical to CDC68, a gene previously shown to be required for passage through the cell cycle control point START. However, at least some transcriptional effects caused by spt16 mutations are independent of arrest at START. These results and those in the accompanying paper (A. Rowley, R. A. Singer, and G. C. Johnston, Mol. Cell. Biol. 11:5718-5726, 1991) indicate that SPT16/CDC68 is required for normal transcription of many loci in S. cerevisiae.

scholarly journals DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4675-4684 ◽  
Author(s):  
F R Cross
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Critical Size ◽  
Size Control ◽  
Mutant Gene ◽  
G1 Phase ◽  
Wild Type ◽  
Alpha Factor ◽  
Multiple Copies ◽  
Kinetics Of

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

Role of transcriptional and posttranscriptional regulation in expression of histone genes in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (2) ◽  
pp. 614-621
Author(s):  
D E Lycan ◽  
M A Osley ◽  
L M Hereford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Posttranscriptional Regulation ◽  
Cell Cycle Control ◽  
Dimensional Structure ◽  
Histone Genes ◽  
Transcriptional Regulatory Mechanisms ◽  
Rna Levels

We analyzed the role of posttranscriptional mechanisms in the regulation of histone gene expression in Saccharomyces cerevisiae. The rapid drop in histone RNA levels associated with the inhibition of ongoing DNA replication was postulated to be due to posttranscriptional degradation of histone transcripts. However, in analyzing the sequences required for this response, we showed that the coupling of histone RNA levels to DNA replication was due mostly, if not entirely, to transcriptional regulatory mechanisms. Furthermore, deletions which removed the negative, cell cycle control sequences from the histone promoter also uncoupled histone transcription from DNA replication. We propose that the arrest of DNA synthesis prematurely activates the regulatory pathway used in the normal cell cycle to repress transcription. Although posttranscriptional regulation did not appear to play a significant role in coupling histone RNA levels to DNA replication, it did affect the levels of histone RNA in the cell cycle. Posttranscriptional regulation could apparently restore much of the periodicity of histone RNA accumulation in cells which constitutively transcribed the histone genes. Unlike transcriptional regulation, periodic posttranscriptional regulation appears to operate on a clock which is independent of events in the mitotic DNA cycle. Posttranscriptional recognition of histone RNA must require either sequences in the 3' end of the RNA or an intact three-dimensional structure since H2A- and H2B-lacZ fusion transcripts, containing only 5' histone sequences, were insensitive to posttranscriptional controls.

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