Cln3-associated kinase activity in Saccharomyces cerevisiae is regulated by the mating factor pathway - PubMed (original) (raw)

Cln3-associated kinase activity in Saccharomyces cerevisiae is regulated by the mating factor pathway

D I Jeoung et al. Mol Cell Biol. 1998 Jan.

Abstract

The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest. Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described. Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest. No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases. Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes. Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest. Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1. Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity. G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance. Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6.

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Figures

FIG. 1

FIG. 1

Regulation of Cln3-associated kinase activity by the mating factor pathway dependent on the Cln3 C terminus. Cells of genotype MATa bar1 GAL1::CLN3C (2928-2B) or GAL1::CLN3-1C (FCMT34-1) (three-HA epitope-tagged version of CLN3 or CLN3-1 coding sequence [56]) and a congenic CLN3 (untagged) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (optical density at 660 nm [OD660] of ∼0.5). α-Factor (αF) was added to 0.5 μM, and incubation continued for 2.5 h. Cells were extracted, and extracts were immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody or anti-Cdc28 antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signal from the untagged control was subtracted from all values before standardizing to the signal from sample 2 (tagged Cln3, no mating factor, set at 100 arbitrary units).

FIG. 2

FIG. 2

C-terminus-dependent regulation of Cln3-associated kinase activity by the mating factor pathway is independent of CLN1 and CLN2. Strains of genotype MATa bar1 GAL1::CLN3C or GAL1::CLN3-1C and a congenic CLN3 (untagged [U]) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). For GAL1::CLN3C and GAL1::CLN3-1C strains, CLN1 CLN2 strains (2928-2B and FCMT34-1) and congenic cln1 cln2 strains (2202-16A and 1807-31B) were tested. α-Factor (αF) was added to 0.5 μM, and incubation continued for 2.5 h. Cells were extracted, and extracts were immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signal from the untagged control was subtracted from all values before standardizing to the signal from sample 3 (tagged Cln3, no mating factor, set at 100 arbitrary units). The amount of Cln3 in the immunoprecipitates was estimated by densitometry of the Western blot signal. The specific activity (S.A.) of the kinase is the kinase recovered divided by the Cln3 Western blot signal, in arbitrary units.

FIG. 3

FIG. 3

Time course and dose response of inhibition of Cln3-associated kinase activity compared to cell cycle arrest. Strains of genotype MATa bar1 GAL1::CLN3C (2928-2B) and a CLN3 (untagged [U]) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). (A) α-Factor (αF) was added to 0.5 μM. At the indicated times, cells were harvested. (B) The indicated concentrations of α-factor were added, and incubation continued for 2.5 h, when cells were harvested. (A and B) The percentage of unbudded cells was determined microscopically. Cells were extracted, and extracts immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signal from the untagged control was subtracted from all values before standardizing to the signal from the time zero sample (tagged Cln3, no mating factor, set at 100 arbitrary units).

FIG. 4

FIG. 4

Regulation of Cln3-associated kinase activity requires a mating factor pathway-regulated MAP kinase. Cells of genotype MATa bar1 GAL1::CLN3C and a CLN3 (untagged) control were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). For both tagged and untagged strains, both FUS3 KSS1 (1255-5C-4b and 1255-5C-4a) and fus3 kss1 (BOY521-3b, -3c, and -3a) strains were tested. α-Factor (αF) was added to 0.5 μM, and incubation continued for 2.5 h. Cells were extracted, and extracts were immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase specific activity (S.A.) was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signals from the untagged controls were subtracted from all values before standardizing to the signal from sample 9 (tagged Cln3, FUS3 KSS1 wild type, no mating factor, set at 100 arbitrary units).

FIG. 5

FIG. 5

Far1 is required for regulation of Cln3-associated kinase specific activity. (A) Cells of genotype MATa bar1 GAL1::CLN3C and a CLN3 (untagged) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). Strains tested were FAR1 (2928-2B) or far1::URA3 (1729-19A). α-Factor (αF) was added to 0.5 μM, and incubation continued for 2.5 h. Cells were extracted, and extracts were immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase specific activity (S.A.) was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signals from the untagged controls were subtracted from all values before standardizing to the signal from sample 2 (tagged Cln3, no mating factor, set at 100 arbitrary units). (B) Cells of genotype MATa bar1 GAL1::CLN3C and a CLN3 (untagged [UT]) control (1729-20D) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). Strains tested were CLN1 CLN2 FAR1 (1729-5C), CLN1 CLN2 far1::URA3 (1729-19A), cln1 cln2 FAR1 (1729-23D), and cln1 cln2 far1::URA3 (1729-11B). α-Factor was added to 0.5 μM, and incubation continued for 2.5 h. Cells were extracted and extracts immunoprecipitated with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signals from the untagged controls were subtracted from all values before standardizing to the signal from sample 2 (tagged Cln3, FAR1 CLN1 CLN2 strain, no mating factor, set at 100 arbitrary units). (C) Cells of genotype MATa bar1 GAL1::CLN3C and a CLN3 (untagged) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). Strains tested were cln1 cln2 FAR1 (1729-23D) and cln1 cln2 far1::URA3 (1729-11B). α-Factor was added to various concentrations (0, 0.001, 0.01, 0.1, and 0.5 μM), and incubation continued for 2.5 h. Cells were extracted, and extracts were immunoprecipitated with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signal from the untagged control was subtracted from all values before standardizing to the signal from sample 3 (tagged Cln3, no mating factor, set at 100 arbitrary units). In panels B and C, the amount of Cln3 in the immunoprecipitates was estimated by densitometry of the Western blot signal. The specific activity (S.A.) of the kinase is the kinase recovered divided by the Cln3 Western blot signal, in arbitrary units.

FIG. 6

FIG. 6

FAR1 overexpression restores mating factor regulation of Cln3-1-associated kinase activity. (A) Cells of genotype MATa bar1 GAL1::CLN3C (2928-2B) or GAL1::CLN3-1C (three-HA epitope-tagged version CLN3-1 coding sequence [56]) (FCMT34-1, FCMT34-2, 1723-6C) and a CLN3 (untagged [U]) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). One GAL1::CLN3-1C strain (1723-6C) also expressed FAR1 from the GAL1 promoter. α-Factor (αF) was added to 0.5 μM, and incubation continued for 2.5 h. Cells were extracted, and extracts were immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody or anti-Far1 antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signals from the untagged controls were subtracted from all values before standardizing to the signal from sample 3 (tagged Cln3, no mating factor, set at 100 arbitrary units). (B) Strain 1723-6C (MATa bar1 GAL1::CLN3-1C GAL1::FAR1) was incubated for 2.5 h in 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, or 1 μM α-factor. Cln3-1 was immunoprecipitated, and H1 kinase activity and coimmunoprecipitated Far1 were assayed as for panel A.

FIG. 7

FIG. 7

FAR1 is required for cell cycle arrest in G1 in cln1 cln2 GAL1::CLN3 strains. Cells of genotype cln1 cln2 cln3 GAL1::CLN3 that were either FAR1 (BOY747) or far1::URA3 (BOY901) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). Cells were collected by filtration, resuspended in YEP-raffinose medium, incubated for 2.5 h at 30°C, and then released from the G1 block by addition of galactose to 3% as described previously (31). Ten minutes before galactose addition, 0.5 μM α-factor (αF) was added to one half of each culture. DNA content was analyzed by flow cytometry, and Northern blot analysis was performed, probing for the indicated transcripts. Open circles, FAR1 culture; closed circles, far1::URA3 culture.

FIG. 8

FIG. 8

G2/M-arrested cells are unable to regulate Cln3-associated kinase activity due to a deficit in Far1 protein abundance. (A) Cells of genotype MATa bar1 GAL1::CLN3C (2928-2B) and a CLN3 (untagged) control (1255-5C) were grown in YEP-Gal medium overnight at 30°C to log phase (OD660 of ∼0.5). Nocodazole (NZ; 15 μg/ml) was added to half of each culture, and incubation continued for 2.5 h to allow mitotic arrest. α-Factor (αF) was added to 0.5 μM, and incubation continued. Cells were extracted, and extracts were immunoprecipitated (IP) with antibody against the epitope tag. Immunoprecipitates were immunoblotted with anti-HA antibody. Cln3-associated histone H1 kinase activity was determined and quantitated with a PhosphorImager. The background H1 phosphorylation signals from the untagged controls were subtracted from all values before standardizing to the signal from sample 2 (tagged Cln3, no mating factor, set at 100 arbitrary units). Lanes 1, 2, 7, and 8 are from untagged cells at 0 and 90 min of α-factor treatment. Other lanes are from tagged cells at 0 (lanes 3 and 9), 30 (lanes 4 and 10), 60 (lanes 5 and 11), and 90 min of α-factor treatment. (B) The experiment was identical to that in panel A except that tagged and untagged strains (1808-2 and 1808-3) contained GAL1::FAR1Δ30 (32) and were incubated in α-factor for 30 min after 2.5 h of nocodazole treatment.

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