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Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase
FEMS Microbiology Letters 153 (1997) 233^236
Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase Hiro-aki Fujimura Central Research Laboratory, Discovery Research Laboratories, Nippon Hoechst Marion Roussel, 1-3-2 Minamidai, Kawagoe 350-11, Japan
Received 21 April 1997 ; revised 30 May 1997; accepted 2 June 1997
Abstract
Tyrphostins are inhibitors of the epidermal growth factor receptor tyrosine kinase. To elucidate the biological function of protein tyrosine kinases in yeast cells, a mutant hypersensitive to tyrphostin was isolated and investigated for its response to the drug. The mutation was recessive and was designated tpt1 for tyrphostin hypersensitive. A tpt1 strain cannot grow in the presence of tyrphostin, implying that a biological process sensitive to tyrphostin is essential for cell growth. Microscopic observation indicated that large-budded cells were accumulated in the presence of the inhibitor. The results suggest the involvement of protein tyrosine phosphorylation in the cell cycle progression of Saccharomyces cerevisiae. Keywords :
Protein tyrosine kinase; Tyrphostin; Cell cycle;
Saccharomyces cerevisiae
1. Introduction
The regulation of protein tyrosine phosphorylation plays an important role in the control of cell growth, di¡erentiation and signalling processes in multicellular organisms [1]. Receptor protein tyrosine kinases have been found in many multicellular organisms and have been extensively studied [2]. Although numerous non-receptor type tyrosine kinases have also been found and analyzed, the function of this modi¢cation of target proteins and the regulation of protein kinase activity in the control of the biological process are poorly understood except for Src kinases [3]. A system which is amenable to genetic analysis would be of bene¢t to the study of tyrosine modi¢cation which is controlled at molecular level. Tyrphostins were developed as drugs which inhibit
epidermal growth factor (EGF) receptor tyrosine kinase activity and have been used as a tool to analyze the biological roles of protein tyrosine phosphorylation in eukaryotic organisms [4^7]. In the yeasts, where the cell cycle has been extensively studied, identi¢cation of the genes involved in the regulation of tyrosine-speci¢c phosphorylation has not been reported, although it has been reported that serine, threonine and tyrosine modi¢cation is genetically controlled [8^11]. To facilitate a genetic study of v-Src function, v-Src was expressed under the control of the GAL1 promoter and large-budded viable cells were found to be accumulated with elevation of p34CDC28 kinase activity in Saccharomyces cerevisiae [12]. In this study I describe the isolation of an S. cerevisiae mutant hypersensitive to tyrphostin which may be useful in the investigation of the biological
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 6 7 - X
FEMSLE 7695 20-10-97
234
H. Fujimura / FEMS Microbiology Letters 153 (1997) 233^236
functions of tyrosine kinases in the regulation of cell proliferation in eukaryotic organisms. 2. Materials and methods
2.1. Yeast strains and media
XF91-5D ( ), LFB5 ( ) and DH14-2C ( ) were used in this study. YEPD (2% glucose, 2% peptone, 1% yeast extract) rich medium and SD (2% glucose, 0.67% yeast nitrogen base without amino acids) minimal medium supplemented with appropriate nutrients were used for cultivation of yeast cells. Growth and manipulation were performed according to standard methods [13]. Tyrphostin was purchased from Sigma Chemicals and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/ml. S. cerevisiae MATa ura3 leu2 his4 trp1 gal2 MATa ura3 leu2 his4 trp1 gal2 tpt1-1 MATa ura3 leu2 met1 tpt1-1
2.2. Isolation of a mutant hypersensitive to tyrphostin
Strain XF91-5D was cultured in YEPD and then mutagenized with 3% ethyl methanesulfonate (EMS) for 1 h at 30³C as described previously [14]. The cells were spread on YEPD plates and replica-plated on SD supplemented with appropriate nutrients with or without tyrphostin (50 Wg/ml). After 3 days of incu-
Fig. 2. Sensitivity of the tpt1 mutant to cycloheximide. The wildtype strain XF91-5D (A, C) and tpt1 mutant LFB5 (B, D) were transferred to YEPD with (C, D) or without(A, B) cycloheximide (0.1 Wg/ml) and incubated for 3 days at room temperature, followed by photography.
bation at 30³C, hypersensitive clones were identi¢ed as those failing to grow in the presence of the inhibitor. 3. Results
3.1. Isolation of mutants
To obtain mutants hypersensitive to tyrphostin, strain XF91-5D was mutagenized with EMS (50% for survival rate). After mutation ¢xation, about 104 colonies were replica-plated on tyrphostin-containing plates and tested for their sensitivity, and a clone was obtained as a hypersensitive strain (Fig. 1). The strain carrying this mutation was designated LFB5 and crossed with the wild-type strain. Tetrad analysis indicated that the hypersensitivity was controlled by a single recessive mutation designated tpt1 for tyrphostin hypersensitive. 3.2. Characterization of the tpt1 mutation
Fig. 1. Sensitivity of the tpt1 mutant to tyrphostin. The parent strain XF91-5D (A, C) and tpt1 mutant LFB5 (B, D) were transferred to a SD minimal plate with (C, D) or without (A, B) tyrphostin (50 Wg/ml) and incubated for 4 days at room temperature, followed by photography.
Yeast cells have many multidrug-resistant genes which control the sensitivity to various types of drugs and metal ions. To clarify whether the tpt1 mutation is related to any multidrug-resistant mutation, the tpt1 mutant was examined for sensitivity to cycloheximide. As shown in Fig. 2, LFB5 has sensi-
FEMSLE 7695 20-10-97
H. Fujimura / FEMS Microbiology Letters 153 (1997) 233^236
235
On the other hand, it was reported that expression of v-Src resulted in accumulation of large-budded cells of
S. cerevisiae
[12]. The large-budded pheno-
type of the v-Src-arrested cells suggested that the block in cell division occurred after START and before cell separation. Cdc28 kinase activity was increased when v-Src was expressed. The yeast cell cycle was arrested in the presence of tyrphostin in
tpt1
cells. Since tyrphostin is believed
Fig. 3. Arrest of the yeast cell cycle by tyrphostin. A culture of
to inhibit the protein tyrosine phosphorylation, the
strain DH14-2C was grown to early exponential phase in SD+9
present result suggests the presence of tyrphostin-
medium and inoculated into SD+9 (A) containing DMSO or (B) containing tyrphostin
(50
Wg/ml).
After 5 h
of incubation at
30³C, samples were taken for microphotography.
S. cerevisiae. However, S. cerevisiae has a Src fam-
sensitive protein kinase in there is no evidence that
CDC28
ily kinase, although p34
has some similarity
with Src kinase. Since the tyrosine phosphorylation tivity to cycloheximide comparable to that of the
of Cdc28 at residue Tyr19 is not crucial for normal
parent strain XF91-5D, in contrast to its tyrphostin
cell division [11], it is unlikely that tyrphostin ar-
sensitivity, indicating that the
rested growth due to inhibition of the Cdc28 protein
cally a¡ects the
tpt1 mutation speci¢sensitivity of S. cerevisiae cells to unlikely that the TPT1 gene is a
kinase. It is possible that tyrphostin inhibits a kinase
tyrphostin. It is
member of the multidrug-resistant genes.
which phosphorylates a protein kinase that in turn CDC28 kinase. phosphorylates p34
3.3. Block of the yeast cell cycle by tyrphostin
yeast cell cycle remains unclear. Further investiga-
The mechanism by which tyrphostin blocks the
tion, especially molecular cloning and analysis of Since the
tpt1
mutant is hypersensitive to tyrphos-
the
TPT1
gene, will give insight into the tyrphos-
tin and cannot grow in the presence of this com-
tin-sensitive protein tyrosine kinase responsible for
pound, the e¡ect of tyrphostin on the cell cycle
progression of the cell cycle.
was investigated. A hypersensitive segregant DH14-
Moreover, a functional screening system was re-
2C, which was more sensitive than the parental mu-
cently developed to identify regulators and antago-
tant strain LFB5, was constructed by tetrad analysis
nizers of protein tyrosine kinases using
and its response to tyrphostin was analyzed. As shown in Fig. 3, the
tpt1
cells did not divide in the
presence of tyrphostin (50
Wg/ml),
and unseparated
Schizosaccharomyces pombe where c-Src was expressed [16]. The present tpt1 mutant may provide a system which allows isolation of inhibitors of protein kinases.
large-budded cells were accumulated.
References 4. Discussion [1] Hanks, S.K., Quinn, A.M. and Hunter, T. (1988) The protein
In this study I have obtained a yeast mutant hypersensitive to an inhibitor of EGF receptor protein tyrosine kinase (tyrphostin), and found that tyrphostin blocks the yeast cell cycle.
the catalytic domains. Science 241, 42^52. [2] Geer, P., Hunter, T. and Lindberg, R.A. (1994) Receptor protein- tyrosine kinases and their signal transduction pathways. Annu. Rev. Cell Biol. 10, 251^337.
Roche et al. reported that a neutralizing antibody speci¢c for Src, Fyn, and Yes inhibited the transition from the G2 phase to mitosis in the cell cycle of mammalian ¢broblasts, and thus indicated the involvement of Src family kinases in the G2-M transition [15].
kinase family : conserved features and deduced phylogeny of
[3] Defranco,
A.L.
(1992)
Tyrosine
phosphorylation
and
the
mechanism of signal transduction by the B-lymphocyte antigen receptor. Eur. J. Biochem. 210, 381^388. [4] Leyton, L., LeGuen, P., Bunch, D. and Saling, P.M. (1992) Regulation of mouse gamete interaction by a sperm tyrosine kinase. Proc. Natl. Acad. Sci. USA 89, 11692^11695. [5] Holen, I., Stromhaug, P.E., Gordon, P.B., Fengsrud, M.,
FEMSLE 7695 20-10-97
H. Fujimura / FEMS Microbiology Letters 153 (1997) 233^236
236
Berg, T.O. and Seglen, P.O. (1995) Inhibition of autophagy and multiple steps in asialoglycoprotein endocytosis by inhibitors of tyrosine protein kinases (tyrphostins). J. Biol. Chem.
rylates proteins on serine, threonine, and tyrosine. Mol. Cell. Biol. 11, 987^1001. [11] Booher, R.N., Deshaies, R.J. and Kirschner, M.W. (1993)
Saccharomyces cerevisiae wee1 and its di¡erent CDC28 regulation of p34 in response to G1 and G2 cyclins. Properties of
270, 12823^12831. [6] Levitzki, A. and Gazit, A. (1995) Tyrosine kinase inhibition : an approach to drug development. Science 267, 1782^ 1788.
EMBO J. 12, 3417^3426. [12] Boschelli, F. (1993) Expression of p60
[7] Akarasereenont, P. and Thiemermann, C. (1996) The induction of cyclo-oxygenase-2 in human pulmonary epithelial cell culture (A549) activated by IL-1
L is inhibited by tyrosine
kin-
ase inhibitors. Biochem. Biophys. Res. Commun. 220, 181^ 185.
cerevisiae
results in elevation of p34
vÿsrc
CDC28
in
Saccharomyces
kinase activity and
release of the dependence of DNA replication on mitosis. Mol. Cell. Biol. 13, 5112^5121. [13] Sherman, F., Fink, G.R. and Hicks, J.B. (1984) Methods in Yeast Genetics. A Laboratory Manual. Cold Spring Harbor
[8] Dailey, D., Schieven, G.L., Lim, M.Y., Marquardt, H., Gil-
Laboratory, Cold Spring Harbor, NY.
more, T., Thorner, J. and Martin, G.S. (1990) Novel yeast
[14] Fujimura, H. (1989) The yeast G-protein homolog is involved
protein kinase (YPK1 gene product) is a 40-kilodalton phos-
in the mating pheromone signal transduction system. Mol.
photyrosyl protein associated with protein-tyrosine kinase activity. Mol. Cell. Biol. 10, 6244^6256. [9] Ballard, M.J., Tyndall, W.A., Shingle, J.M., Hall, D.J. and Winter, E. (1991) Tyrosine phosphorylation of a yeast 40 kDa
protein
occurs
in
response
to
mating
pheromone.
EMBO J. 10, 3753^3758.
quirement for Src family protein tyrosine kinases in G2 for ¢broblast cell division. Science 269, 1567^1569. [16] Superti-Furga, G., Joensson, K. and Courtneidge, S.A. (1996) A functional screen in yeast for regulators and antagonizers of
[10] Stern, D.F., Zheng, P., Beidler, D.R. and Zerillo, C. (1991) SPK1, a new kinase from
Cell. Biol. 9, 152^158. [15] Roche, S., Fumagalli, S. and Courtneidge, S.A. (1995) Re-
Saccharomyces cerevisiae,
phospho-
heterologous protein tyrosine kinases. Nature Biotechnol. 14, 600^605.
FEMSLE 7695 20-10-97
Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase Hiro-aki Fujimura Central Research Laboratory, Discovery Research Laboratories, Nippon Hoechst Marion Roussel, 1-3-2 Minamidai, Kawagoe 350-11, Japan
Received 21 April 1997 ; revised 30 May 1997; accepted 2 June 1997
Abstract
Tyrphostins are inhibitors of the epidermal growth factor receptor tyrosine kinase. To elucidate the biological function of protein tyrosine kinases in yeast cells, a mutant hypersensitive to tyrphostin was isolated and investigated for its response to the drug. The mutation was recessive and was designated tpt1 for tyrphostin hypersensitive. A tpt1 strain cannot grow in the presence of tyrphostin, implying that a biological process sensitive to tyrphostin is essential for cell growth. Microscopic observation indicated that large-budded cells were accumulated in the presence of the inhibitor. The results suggest the involvement of protein tyrosine phosphorylation in the cell cycle progression of Saccharomyces cerevisiae. Keywords :
Protein tyrosine kinase; Tyrphostin; Cell cycle;
Saccharomyces cerevisiae
1. Introduction
The regulation of protein tyrosine phosphorylation plays an important role in the control of cell growth, di¡erentiation and signalling processes in multicellular organisms [1]. Receptor protein tyrosine kinases have been found in many multicellular organisms and have been extensively studied [2]. Although numerous non-receptor type tyrosine kinases have also been found and analyzed, the function of this modi¢cation of target proteins and the regulation of protein kinase activity in the control of the biological process are poorly understood except for Src kinases [3]. A system which is amenable to genetic analysis would be of bene¢t to the study of tyrosine modi¢cation which is controlled at molecular level. Tyrphostins were developed as drugs which inhibit
epidermal growth factor (EGF) receptor tyrosine kinase activity and have been used as a tool to analyze the biological roles of protein tyrosine phosphorylation in eukaryotic organisms [4^7]. In the yeasts, where the cell cycle has been extensively studied, identi¢cation of the genes involved in the regulation of tyrosine-speci¢c phosphorylation has not been reported, although it has been reported that serine, threonine and tyrosine modi¢cation is genetically controlled [8^11]. To facilitate a genetic study of v-Src function, v-Src was expressed under the control of the GAL1 promoter and large-budded viable cells were found to be accumulated with elevation of p34CDC28 kinase activity in Saccharomyces cerevisiae [12]. In this study I describe the isolation of an S. cerevisiae mutant hypersensitive to tyrphostin which may be useful in the investigation of the biological
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 6 7 - X
FEMSLE 7695 20-10-97
234
H. Fujimura / FEMS Microbiology Letters 153 (1997) 233^236
functions of tyrosine kinases in the regulation of cell proliferation in eukaryotic organisms. 2. Materials and methods
2.1. Yeast strains and media
XF91-5D ( ), LFB5 ( ) and DH14-2C ( ) were used in this study. YEPD (2% glucose, 2% peptone, 1% yeast extract) rich medium and SD (2% glucose, 0.67% yeast nitrogen base without amino acids) minimal medium supplemented with appropriate nutrients were used for cultivation of yeast cells. Growth and manipulation were performed according to standard methods [13]. Tyrphostin was purchased from Sigma Chemicals and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/ml. S. cerevisiae MATa ura3 leu2 his4 trp1 gal2 MATa ura3 leu2 his4 trp1 gal2 tpt1-1 MATa ura3 leu2 met1 tpt1-1
2.2. Isolation of a mutant hypersensitive to tyrphostin
Strain XF91-5D was cultured in YEPD and then mutagenized with 3% ethyl methanesulfonate (EMS) for 1 h at 30³C as described previously [14]. The cells were spread on YEPD plates and replica-plated on SD supplemented with appropriate nutrients with or without tyrphostin (50 Wg/ml). After 3 days of incu-
Fig. 2. Sensitivity of the tpt1 mutant to cycloheximide. The wildtype strain XF91-5D (A, C) and tpt1 mutant LFB5 (B, D) were transferred to YEPD with (C, D) or without(A, B) cycloheximide (0.1 Wg/ml) and incubated for 3 days at room temperature, followed by photography.
bation at 30³C, hypersensitive clones were identi¢ed as those failing to grow in the presence of the inhibitor. 3. Results
3.1. Isolation of mutants
To obtain mutants hypersensitive to tyrphostin, strain XF91-5D was mutagenized with EMS (50% for survival rate). After mutation ¢xation, about 104 colonies were replica-plated on tyrphostin-containing plates and tested for their sensitivity, and a clone was obtained as a hypersensitive strain (Fig. 1). The strain carrying this mutation was designated LFB5 and crossed with the wild-type strain. Tetrad analysis indicated that the hypersensitivity was controlled by a single recessive mutation designated tpt1 for tyrphostin hypersensitive. 3.2. Characterization of the tpt1 mutation
Fig. 1. Sensitivity of the tpt1 mutant to tyrphostin. The parent strain XF91-5D (A, C) and tpt1 mutant LFB5 (B, D) were transferred to a SD minimal plate with (C, D) or without (A, B) tyrphostin (50 Wg/ml) and incubated for 4 days at room temperature, followed by photography.
Yeast cells have many multidrug-resistant genes which control the sensitivity to various types of drugs and metal ions. To clarify whether the tpt1 mutation is related to any multidrug-resistant mutation, the tpt1 mutant was examined for sensitivity to cycloheximide. As shown in Fig. 2, LFB5 has sensi-
FEMSLE 7695 20-10-97
H. Fujimura / FEMS Microbiology Letters 153 (1997) 233^236
235
On the other hand, it was reported that expression of v-Src resulted in accumulation of large-budded cells of
S. cerevisiae
[12]. The large-budded pheno-
type of the v-Src-arrested cells suggested that the block in cell division occurred after START and before cell separation. Cdc28 kinase activity was increased when v-Src was expressed. The yeast cell cycle was arrested in the presence of tyrphostin in
tpt1
cells. Since tyrphostin is believed
Fig. 3. Arrest of the yeast cell cycle by tyrphostin. A culture of
to inhibit the protein tyrosine phosphorylation, the
strain DH14-2C was grown to early exponential phase in SD+9
present result suggests the presence of tyrphostin-
medium and inoculated into SD+9 (A) containing DMSO or (B) containing tyrphostin
(50
Wg/ml).
After 5 h
of incubation at
30³C, samples were taken for microphotography.
S. cerevisiae. However, S. cerevisiae has a Src fam-
sensitive protein kinase in there is no evidence that
CDC28
ily kinase, although p34
has some similarity
with Src kinase. Since the tyrosine phosphorylation tivity to cycloheximide comparable to that of the
of Cdc28 at residue Tyr19 is not crucial for normal
parent strain XF91-5D, in contrast to its tyrphostin
cell division [11], it is unlikely that tyrphostin ar-
sensitivity, indicating that the
rested growth due to inhibition of the Cdc28 protein
cally a¡ects the
tpt1 mutation speci¢sensitivity of S. cerevisiae cells to unlikely that the TPT1 gene is a
kinase. It is possible that tyrphostin inhibits a kinase
tyrphostin. It is
member of the multidrug-resistant genes.
which phosphorylates a protein kinase that in turn CDC28 kinase. phosphorylates p34
3.3. Block of the yeast cell cycle by tyrphostin
yeast cell cycle remains unclear. Further investiga-
The mechanism by which tyrphostin blocks the
tion, especially molecular cloning and analysis of Since the
tpt1
mutant is hypersensitive to tyrphos-
the
TPT1
gene, will give insight into the tyrphos-
tin and cannot grow in the presence of this com-
tin-sensitive protein tyrosine kinase responsible for
pound, the e¡ect of tyrphostin on the cell cycle
progression of the cell cycle.
was investigated. A hypersensitive segregant DH14-
Moreover, a functional screening system was re-
2C, which was more sensitive than the parental mu-
cently developed to identify regulators and antago-
tant strain LFB5, was constructed by tetrad analysis
nizers of protein tyrosine kinases using
and its response to tyrphostin was analyzed. As shown in Fig. 3, the
tpt1
cells did not divide in the
presence of tyrphostin (50
Wg/ml),
and unseparated
Schizosaccharomyces pombe where c-Src was expressed [16]. The present tpt1 mutant may provide a system which allows isolation of inhibitors of protein kinases.
large-budded cells were accumulated.
References 4. Discussion [1] Hanks, S.K., Quinn, A.M. and Hunter, T. (1988) The protein
In this study I have obtained a yeast mutant hypersensitive to an inhibitor of EGF receptor protein tyrosine kinase (tyrphostin), and found that tyrphostin blocks the yeast cell cycle.
the catalytic domains. Science 241, 42^52. [2] Geer, P., Hunter, T. and Lindberg, R.A. (1994) Receptor protein- tyrosine kinases and their signal transduction pathways. Annu. Rev. Cell Biol. 10, 251^337.
Roche et al. reported that a neutralizing antibody speci¢c for Src, Fyn, and Yes inhibited the transition from the G2 phase to mitosis in the cell cycle of mammalian ¢broblasts, and thus indicated the involvement of Src family kinases in the G2-M transition [15].
kinase family : conserved features and deduced phylogeny of
[3] Defranco,
A.L.
(1992)
Tyrosine
phosphorylation
and
the
mechanism of signal transduction by the B-lymphocyte antigen receptor. Eur. J. Biochem. 210, 381^388. [4] Leyton, L., LeGuen, P., Bunch, D. and Saling, P.M. (1992) Regulation of mouse gamete interaction by a sperm tyrosine kinase. Proc. Natl. Acad. Sci. USA 89, 11692^11695. [5] Holen, I., Stromhaug, P.E., Gordon, P.B., Fengsrud, M.,
FEMSLE 7695 20-10-97
H. Fujimura / FEMS Microbiology Letters 153 (1997) 233^236
236
Berg, T.O. and Seglen, P.O. (1995) Inhibition of autophagy and multiple steps in asialoglycoprotein endocytosis by inhibitors of tyrosine protein kinases (tyrphostins). J. Biol. Chem.
rylates proteins on serine, threonine, and tyrosine. Mol. Cell. Biol. 11, 987^1001. [11] Booher, R.N., Deshaies, R.J. and Kirschner, M.W. (1993)
Saccharomyces cerevisiae wee1 and its di¡erent CDC28 regulation of p34 in response to G1 and G2 cyclins. Properties of
270, 12823^12831. [6] Levitzki, A. and Gazit, A. (1995) Tyrosine kinase inhibition : an approach to drug development. Science 267, 1782^ 1788.
EMBO J. 12, 3417^3426. [12] Boschelli, F. (1993) Expression of p60
[7] Akarasereenont, P. and Thiemermann, C. (1996) The induction of cyclo-oxygenase-2 in human pulmonary epithelial cell culture (A549) activated by IL-1
L is inhibited by tyrosine
kin-
ase inhibitors. Biochem. Biophys. Res. Commun. 220, 181^ 185.
cerevisiae
results in elevation of p34
vÿsrc
CDC28
in
Saccharomyces
kinase activity and
release of the dependence of DNA replication on mitosis. Mol. Cell. Biol. 13, 5112^5121. [13] Sherman, F., Fink, G.R. and Hicks, J.B. (1984) Methods in Yeast Genetics. A Laboratory Manual. Cold Spring Harbor
[8] Dailey, D., Schieven, G.L., Lim, M.Y., Marquardt, H., Gil-
Laboratory, Cold Spring Harbor, NY.
more, T., Thorner, J. and Martin, G.S. (1990) Novel yeast
[14] Fujimura, H. (1989) The yeast G-protein homolog is involved
protein kinase (YPK1 gene product) is a 40-kilodalton phos-
in the mating pheromone signal transduction system. Mol.
photyrosyl protein associated with protein-tyrosine kinase activity. Mol. Cell. Biol. 10, 6244^6256. [9] Ballard, M.J., Tyndall, W.A., Shingle, J.M., Hall, D.J. and Winter, E. (1991) Tyrosine phosphorylation of a yeast 40 kDa
protein
occurs
in
response
to
mating
pheromone.
EMBO J. 10, 3753^3758.
quirement for Src family protein tyrosine kinases in G2 for ¢broblast cell division. Science 269, 1567^1569. [16] Superti-Furga, G., Joensson, K. and Courtneidge, S.A. (1996) A functional screen in yeast for regulators and antagonizers of
[10] Stern, D.F., Zheng, P., Beidler, D.R. and Zerillo, C. (1991) SPK1, a new kinase from
Cell. Biol. 9, 152^158. [15] Roche, S., Fumagalli, S. and Courtneidge, S.A. (1995) Re-
Saccharomyces cerevisiae,
phospho-
heterologous protein tyrosine kinases. Nature Biotechnol. 14, 600^605.
FEMSLE 7695 20-10-97