scholarly journals Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (9) ◽  
pp. 5290-5300 ◽  
Author(s):  
S M Murphy ◽  
M Bergman ◽  
D O Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Kinase Activity ◽  
Src Kinase ◽  
Intact Cells ◽  
Sh3 Domains ◽  
Mutant Proteins ◽  
Dependent Growth

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5290-5300
Author(s):  
S M Murphy ◽  
M Bergman ◽  
D O Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Kinase Activity ◽  
Src Kinase ◽  
Intact Cells ◽  
Sh3 Domains ◽  
Mutant Proteins ◽  
Dependent Growth

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity

1982 ◽  
Vol 2 (10) ◽  
pp. 1187-1198 ◽  
Author(s):  
B S Schaffhausen ◽  
H Dorai ◽  
G Arakere ◽  
T L Benjamin
Keyword(s):  
Kinase Activity ◽  
T Antigen ◽  
Polyoma Virus ◽  
Intact Cells ◽  
Apparent Lack ◽  
Permeabilized Cells ◽  
Kinase Reaction ◽  
Middle T Antigen

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.

scholarly journals Src acts as the target of matrine to inhibit the proliferation of cancer cells by regulating phosphorylation signaling pathways

2020 ◽  
Author(s):  
Xi Zhang ◽  
Hui Xu ◽  
Xiaoyang Bi ◽  
Guoqing Hou ◽  
Andong Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Src Kinase ◽  
Kinase Domain ◽  
Akt Phosphorylation ◽  
In Vivo Experiments

Background and Purpose: Identification of accurate targets is essential for a successful development of targeted therapy in cancer. Studies have shown that matrine has antitumor activity against many types of cancers. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Experimental Approach: The effect of matrine on the proliferation of cancer cells were examined by MTT assay. Pull-down assay and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were performed to explore the target of matrine. A series of in vitro and in vivo experiments were conducted to reveal the mechanisms by which matrine targeted Src to regulate the downstream signaling pathways of Src in cancer cells. Key Results: Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with matrine-amino coupling resins (MA beads) and LC-MS/MS identified Src as the target of matrine. Src kinase domain is required for its interaction with matrine and Ala392 in the kinase domain participated in matrine-Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3 and PI3K/Akt signaling pathways. Conclusions and Implications: Collectively, matrine targeted Src, inhibited kinase activity and down-regulated its downstream MAPK/ERK, JAK2/STAT3 and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells.

scholarly journals Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation

2001 ◽  
Vol 155 (5) ◽  
pp. 763-774 ◽  
Author(s):  
Jung-seog Kang ◽  
Iain M. Cheeseman ◽  
George Kallstrom ◽  
Soundarapandian Velmurugan ◽  
Georjana Barnes ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Related Protein ◽  
Centromeric Dna ◽  
Centromere Protein ◽  
Kinetochore Function

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.

scholarly journals Std1p (Msn3p) Positively Regulates the Snf1 Kinase in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 507-514 ◽  
Author(s):  
Sergei Kuchin ◽  
Valmik K Vyas ◽  
Ellen Kanter ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Snf1 Kinase ◽  
Glucose Signaling ◽  
Catalytic Domains ◽  
Upstream Kinase ◽  
Two Hybrid

Abstract The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

scholarly journals Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2566-2575 ◽  
Author(s):  
T E Blank ◽  
M P Woods ◽  
C M Lebo ◽  
P Xin ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Strong Support ◽  
Constitutive Expression ◽  
Mutant Proteins ◽  
Binding Characteristics ◽  
Isolation And Characterization ◽  
In Cells

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

scholarly journals Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity.

1982 ◽  
Vol 2 (10) ◽  
pp. 1187-1198 ◽  
Author(s):  
B S Schaffhausen ◽  
H Dorai ◽  
G Arakere ◽  
T L Benjamin
Keyword(s):  
Kinase Activity ◽  
T Antigen ◽  
Polyoma Virus ◽  
Intact Cells ◽  
Apparent Lack ◽  
Permeabilized Cells ◽  
Kinase Reaction ◽  
Middle T Antigen

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.

Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro

2003 ◽  
Vol 285 (5) ◽  
pp. G861-G869 ◽  
Author(s):  
Douglas A. Potoka ◽  
Jeffrey S. Upperman ◽  
Xiao-Ru Zhang ◽  
Joshua R. Kaplan ◽  
Seth J. Corey ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Src Kinase ◽  
Dependent Manner ◽  
Gut Barrier ◽  
Enterocyte Proliferation ◽  
Barrier Failure ◽  
Enterocyte Apoptosis ◽  
Gut Barrier Failure

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO-may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO-on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO-or decomposed ONOO-in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2′-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO-treatment in vitro inhibited enterocyte proliferation. ONOO-treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO-blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO-may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.

scholarly journals Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro

2008 ◽  
Vol 8 (3) ◽  
pp. 306-314 ◽  
Author(s):  
Régine Bosson ◽  
Isabelle Guillas ◽  
Christine Vionnet ◽  
Carole Roubaty ◽  
Andreas Conzelmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
In Vitro Assay ◽  
Gpi Anchor ◽  
Intact Cells ◽  
Vitro Assay ◽  
Gpi Proteins ◽  
Mutant Cells

ABSTRACT After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [3H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [3H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [3H]DHS into the same abnormal anchor lipids as gup1Δ cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [3H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C26:0-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [3H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.

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