Mechanisms of growth-dependent regulation of the Gin4 kinase

Abstract

Cell cycle progression is dependent upon cell growth. Cells must therefore translate growth into a proportional signal that can be used to determine when there has been sufficient growth for cell cycle progression. In budding yeast, the protein kinase Gin4 is required for normal control of cell growth and undergoes gradual hyperphosphorylation and activation that are dependent upon growth and proportional to the extent of growth, which suggests that Gin4 could function in mechanisms that measure cell growth. However, the molecular mechanisms that drive hyperphosphorylation of Gin4 are poorly understood. Here, we used biochemical reconstitution and genetic analysis to test hypotheses for the mechanisms that drive phosphorylation of Gin4. We ruled out a previous model in which phosphatidylserine delivered to sites of plasma membrane growth binds Gin4 to initiate autophosphorylation. Instead, we show that Elm1, a homolog of the mammalian Lkb1 tumor suppressor kinase, is sufficient to promote hyperphosphorylation of Gin4 in vitro, likely via initiation of Gin4 autophosphorylation. Furthermore, we show that casein kinase I is required for growth-dependent hyperphosphorylation of Gin4 and also for normal regulation of Elm1. Together, these discoveries lead to new insight into mechanisms that link cell cycle progression to cell growth.

Figure 1.. Binding of phosphatidylserine is not sufficient to induce full hyperphosphorylation of Gin4

A) Purification of Gin4-MBP-8xHis from log-phase yeast cells. Lane 1: cell extract, lane 2: nickel column flow-through, lane 3: nickel column elution, lane 4: amylose column flow-through, lane 5: amylose column elution. A lower molecular-weight band that is detected with an anti-Gin4 antibody and a likely Gin4 breakdown product is labeled. An anti-Gin4 western blot of the samples shown in the Coomassie blue-stained gel is shown below. B) Western blot analysis of Gin4 phosphorylation. Lanes 1 and 2 show the behavior of endogenous Gin4 in extracts from cells arrested in interphase or mitosis. Lanes 3 and 4 show purified Gin4, pre-treated with TEV protease at 30°C for 45 minutes, at 60 nM in the absence and presence of ATP. Lanes 5–9 show the effects of adding phosphatidylserine. Phosphatidylserine was added as lipid vesicles containing phosphatidylcholine and 15%−30% (mol%) phosphatidylserine. The final concentration of phosphatidylserine ranged from 0.075 uM to 0.15 uM. All reactions were carried out at 30°C for 45 minutes.

Figure 2.. Purified Elm1 can induce full hyperphosphorylation of Gin4 in vitro.

A) Coomassie blue-stained gel showing the purified Elm1 kinase used for in vitro kinase assays. B) Purified Gin4 kinase at 60 nM was pre-cleaved with TEV protease at 30°C to remove the MBP-8XHIS tag and incubated with ATP alone, and with ATP and increasing concentrations of purified Elm1 kinase at 30°C for 45 minutes. Gin4 phosphorylation was assayed by electrophoretic mobility gel shift via western blotting. C) Purified wild type and kinase dead Gin4 kinase were incubated with Elm1 kinase in the presence of ATP at 30°C for 45 minutes. Gin4 phosphorylation was assayed by electrophoretic mobility gel shift via western blot. D) Cells that contain Gin4-GST and Gin4–3XHA were arrested in mitosis with benomyl and Gin4-GST was immunoprecipitated with anti-GST antibody. As a control, immunoprecipitations were also carried out with a non-specific antibody (anti-MBP). Total lysates and immunoprecipitations were probed with anti-HA to detect Gin4–3XHA.

Figure 3.. Yck1/2 kinase activity is required for Gin4 hyperphosphorylation and normal mitotic progression in vivo.

Wild type and yck1Δ yck2-as cells were released from a G1 phase arrest in YPD medium without supplemental adenine at room temperature and 3-MOB-PP1 analog was added 15 minutes after release from arrest. Samples were collected at the indicated times and analyzed by western blot. An anti-Nap1 antibody was used for a loading control.

Figure 4.. Yck1/2 localization to the plasma membrane is required for Gin4 hyperphosphorylation and normal mitotic progression in vivo.

Wild type and akr1Δ cells were released from a G1 phase arrest in YPD medium at room temperature. Samples were collected at the indicated times and analyzed by western blot. An anti-Nap1 antibody was used for a loading control.

Figure 5.. Yck1/2 are required for normal regulation of Elm1.

A) Elm1–9XMyc cells were lysed in the presence of phosphatase inhibitors or in the presence of lambda phosphatase. Elm1 phosphorylation was assayed by western blot to detect electrophoretic mobility shifts. B) Wild type cells and yck1Δ yck2-as cells that express Elm1–9XMyc from the endogenous locus were released from a G1 phase arrest into YPD medium without supplemental adenine at room temperature and treated with 3-MOB-PP1 analog 30 minutes after release from the arrest. Samples were collected at the indicated times and analyzed by western blot to detect Elm1–9XMyc. C) Purified Elm1 at 200 nM was incubated with ATP alone or with ATP and purified Yck1 at 600 nM at 30°C for 45 minutes. Elm1 phosphorylation was assayed by electrophoretic mobility shift via western blot.