Yeast glycogen synthase kinase 3 is involved in protein degradation in cooperation with Bul1, Bul2, and Rsp5

Abstract

The yeast Saccharomyces cerevisiae has four genes, MCK1, MDS1 (RIM11), MRK1, and YOL128c, that encode glycogen synthase kinase 3 (GSK-3) homologs. The gsk-3 null mutant, in which these four genes are disrupted, shows temperature sensitivity, which is suppressed by the expression of mammalian GSK-3beta and by an osmotic stabilizer. Suppression of temperature sensitivity by an osmotic stabilizer is also observed in the bul1 bul2 double null mutant, and the temperature sensitivity of the bul1 bul2 double null mutant is suppressed by multiple copies of MCK1. We have screened rog mutants (revertants of gsk-3) which suppress the temperature sensitivity of the mck1 mds1 double null mutant and found that two of them, rog1 and rog2, also suppress the temperature sensitivity of the bul1 bul2 double null mutant. Bul1 and Bul2 have been reported to bind to Rsp5, a hect (for homologous to E6-associated-protein carboxyl terminus)-type ubiquitin ligase, but involvement of Bul1 and Bul2 in protein degradation has not been demonstrated. We find that Rog1, but not Rog2, is stabilized in the gsk-3 null and the bul1 bul2 double null mutants. Rog1 binds directly to Rsp5, and their interaction is dependent on GSK-3. Furthermore, Rog1 is stabilized in the npi1 mutant, in which RSP5 expression levels are reduced. These results suggest that yeast GSK-3 regulates the stability of Rog1 in cooperation with Bul1, Bul2, and Rsp5.

FIG. 1

Suppression of the temperature sensitivity of the gsk-3 null mutant by an osmotic stabilizer and expression of mammalian GSK-3β. (A) The strains W303a (wild type [WT]) and YTA003W (Δgsk-3) were streaked on YPD plates (b and c) or a plate containing YPD plus 1.2 M sorbitol (d) as indicated in panel a and incubated at 30°C (b) or 37°C (c and d) for 3 days. (B) YTA003W was transformed with pKT10 vector, pTA024 (pKT10-GSK3β), and pTA021 (YCp50-MCK1), streaked on an SC-Ura plate, and incubated at 37°C for 3 days.

FIG. 2

Suppression of the temperature sensitivity of the bul1 bul2 double null mutant by multiple copies of MCK1. Strain YHY009K (Δbul1 Δbul2) was transformed with YEp24 vector, pTA022 (YEp24-MCK1), and pHY06 (YCp-BUL1), streaked on an SC-Ura plate, and incubated at 37°C for 3 days.

FIG. 3

Suppression of the temperature sensitivities of both the bul1 bul2 double null mutant and the mck1 mds1 double null mutant by rog1 (A and B); temperature sensitivity conferred by overexpression of ROG1 (C). (A) KA31a (wild type [WT]), YTA002K (Δmck1 Δmds1), and YTA004K (rog1 Δmck1 Δmds1) were streaked on a YPD plate and incubated at 37°C for 3 days. (B) KA31a (WT), YHY009K (Δbul1 Δbul2), and YTA101K (rog1 Δbul1 Δbul2) were streaked on a YPD plate and incubated at 37°C for 3 days. (C) KA31a was transformed with pKT10 or pTA028 (pKT10-myc-ROG1), streaked on an SC-Ura plate, and incubated at 37°C for 3 days.

FIG. 4

Dependence of Rog1 degradation on Bul1 and Bul2 and GSK-3. (A) Growth curves of strains W303a (wild type [WT]), YTA003W (Δgsk-3), KA31a (WT), and YHY009K (Δbul1 Δbul2) carrying pTA028 (pKT10-myc-Rog1). Exponentially growing cells in MV medium at 30°C were shifted to 37°C at 0 h, and OD₆₀₀ was measured at the indicated times. W303a (WT), YTA003W (Δgsk-3), KA31a (WT), and YHY009K (Δbul1 Δbul2) carrying pTA028 (pKT10-myc-Rog1) (B and C) or pTA030 (pKT10-myc-Rog2) (D and E) were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine for 20 min and chased by cold methionine and cysteine for 0, 1, 2, and 4 h. Cells were lysed and myc-Rog1 was immunoprecipitated with the anti-myc antibody, followed by radioluminography (B and D). The relative amounts of ³⁵S-labeled proteins at each of the times in panels B and D were expressed as the percentages of ³⁵S-labeled myc-Rog1 at time zero in panels C and E, respectively. The results shown are the representative results (B and D) or the means ± standard errors (C and E) of three independent experiments. (F) Exponentially growing cells with an OD₆₀₀ of 0.5 of strains W303a (wild type [WT]), YTA003W (Δgsk-3), KA31a (WT), and YHY009K (Δbul1 Δbul2) carrying pTA026 (pRS316-GAL-HA-Rog1) in SG-Ura at 30°C were harvested, and the whole lysates were probed with the anti-HA antibody.

FIG. 5

Complex formation of Rog1, Rsp5, and Bul1. (A) Binding of Rog1 to Rsp5 in vitro. (a) MBP-Rsp5 and GST-Rog1 purified from E. coli were subjected to gel electrophoresis followed by Coomassie brilliant blue staining. The arrows show the positions of MBP-Rsp5 and GST-Rog1. (b) The purified GST-Rog1 was incubated with MBP-Rsp5 or MBP immobilized to amylose resin at 4°C for 1 h, and the precipitates were probed with the anti-GST antibody. (B) Binding of Rog1 to Rsp5 in intact cells. (a) Cell lysates of W303a (wild type [WT]) cells carrying both pHY22 (myc-Rsp5) and pTA026 (HA-Rog1) or one of the two were directly probed with or immunoprecipitated (IP) with the anti-myc or the anti-HA antibody. The immunoprecipitates were probed with the same antibodies. (b) Cell lysates of W303a (WT) carrying both pHY20 (HA-Bul1) and pTA025 (myc-Rog1) or pHY20 alone were directly probed with the anti-myc and anti-HA antibodies or immunoprecipitated with the anti-myc antibody. The immunoprecipitates were probed with the anti-myc and anti-HA antibodies.

FIG. 6

Degradation of Rog1 in the npi1 mutant and suppression of the temperature sensitivity of the rsp5-101 mutant by rog1. (A) Pulse-chase analyses of myc-Rog1 were performed as described in the legend for Fig. 4. Pulse-labeled myc-Rog1 in strain 24346c (wild type [WT]) or 27038a (npi1) carrying pTA028 (pKT10-myc-Rog1) was immunoprecipitated and subjected to radioluminography. (B) The relative amounts of radiolabeled myc-Rog1 at each of the times in panel A were expressed as the percentages of ³⁵S-labeled myc-Rog1 at time zero. The results shown are the representative results (A) or the means ± standard errors (B) of two independent experiments. (C) KA31a (WT), YTA2-1C (rsp5-101), and YTA102K (rog1 rsp5-101) cells were streaked on a YPD plate and incubated at 35°C for 2 days.

FIG. 7

Regulation of the complex formation of Rog1 with Rsp5 by GSK-3. (A) Lysates from cells carrying pHY22 (myc-Rsp5) and cells transformed with pTA026 (HA-Rog1) (wild type [WT] and Δgsk-3) or transformed with pRS316-GAL-HA-BS vector (control) were prepared. As the amount of HA-Rog1 in the gsk-3 null mutant was more than that in wild-type cells, the levels of HA-Rog1 in the lysates of both cells were normalized. The lysates expressing myc-Rsp5 and HA-Rog1 individually from the wild-type or the gsk-3 null cells were mixed and the aliquots were probed with the anti-myc and anti-HA antibodies. The mixture of the lysates was immunoprecipitated (IP) with the anti-HA antibody, and then the immunoprecipitates were probed with the anti-myc and anti-HA antibodies. (B) In vivo phosphorylation of Rog1. W303a (WT), YTA003W (Δgsk-3), KA31a (WT), and YHY009K (Δbul1 Δbul2) carrying pTA028 (pKT10-myc-Rog1) were labeled with ³²P_i at 37°C for 3 h, and the normalized amount of myc-Rog1 (lower panel) was subjected to autoradiography (upper panel). The results shown are representative of two independent experiments.

FIG. 8

Suppression of the growth defects of the bul1 bul2 double null mutant on nonfermentable carbon sources by rog1. KA31a (wild type [WT]), YHY009K (Δbul1 Δbul2), and YTA101K (rog1 Δbul1 Δbul2) were streaked on YPD, YPGlycerol, YPEthanol, and YPAcetate plates as indicated in panel A and incubated at 26°C for 2 days (B and C) or 4 days (D and E).

FIG. 9

Possible model of functional interaction between GSK-3, Bul1 and Bul2, and Rsp5. (A) Usually, GSK-3 promotes dephosphorylation of Rog1, which is recognized by Bul1 and Bul2 and Rsp5, resulting in the degradation of Rog1. (B and C) Both the phosphorylated form of Rog1, which accumulates in the gsk-3 null mutant (B), and the dephosphorylated form of Rog1, which accumulates in the bul1 bul2 double null or the rsp5 mutant (C), may inhibit cell growth at 37°C.