Cell cycle-dependent assembly of a Gin4-septin complex - PubMed (original) (raw)

Cell cycle-dependent assembly of a Gin4-septin complex

Eric M Mortensen et al. Mol Biol Cell. 2002 Jun.

Abstract

Gin4, a Nim1-related kinase, is required in budding yeast for localization of the septins and for proper control of daughter cell growth during G2/M. Gin4 becomes hyperphosphorylated when cells enter mitosis, leading to activation of Gin4 kinase activity. In this study, we have used immunoaffinity chromatography to identify proteins that associate with Gin4 during mitosis, with the goal of finding targets of Gin4 kinase activity and proteins that play a role in Gin4 activation. We show that during mitosis Gin4 is assembled into a multiprotein complex that includes Nap1, Bni5, the septins, and at least two molecules of Gin4. The associated Gin4 molecules present in this complex phosphorylate each other, leading to Gin4 hyperphosphorylation. Furthermore, the Shs1 septin present in the complex undergoes Gin4-dependent phosphorylation during mitosis and appears to be a substrate of Gin4 in vitro, suggesting that it is a target of Gin4 kinase activity in vivo. Genetic data support the idea that Shs1 is an important target of Gin4 kinase activity. Association of Gin4 with the septins during mitosis requires Shs1, Nap1, Cla4, Elm1, and the kinase activities of Gin4 and Cdc28. Self-association of Gin4 molecules requires Shs1 but not Cla4 or Nap1. Previous work has suggested that the septins function together as a tight complex, and we found that the majority of the Shs1 in the cell is tightly bound to the other septins Cdc3, Cdc10, Cdc11, and Cdc12. Interestingly, however, Shs1 can bind to Gin4 and induce Gin4 oligomerization under conditions in which the Cdc11 septin does not bind to Gin4, suggesting that Shs1 can function independently of the other septins. Taken together, these findings suggest that highly regulated protein-binding events ensure that the Gin4 kinase is activated only during mitosis and only in association with Shs1, a likely in vivo substrate of Gin4. In addition, these results provide clues to how Gin4 may regulate the localization or function of the septins.

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Figures

Figure 1

Figure 1

Gin4 autophosphorylation is due to an intermolecular event. Haploid or diploid cells expressing 3×HA-tagged versions of wild-type GIN4 and/or a catalytically inactive GIN4 (gin4 K48A) were arrested in mitosis with the microtubule-depolymerizing drug benomyl, and the electrophoretic mobility of 3×HA-GIN4 was monitored by Western blotting with an anti-HA antibody. A strain carrying wild-type_3×HA-GIN4_ arrested in G1 with α factor is included for comparison. The genotype of each strain is indicated at the top of the figure, and the cell cycle arrest point is indicated below (I, interphase; M, mitosis).

Figure 2

Figure 2

Gin4 oligomerization is mitosis specific. A strain carrying 3×HA-GIN4 and GST-GIN4 (strain EM9) was arrested in interphase or mitosis, and GST-GIN4 was immunoprecipitated with anti-GST antibodies. The coprecipitation of 3×HA-Gin4 was detected by Western blotting of the precipitates with anti-HA antibody. A control immunoprecipitation (IP) was carried out using equal amounts of a nonspecific antibody. Western blotting of the crude extracts revealed that 3×HA-Gin4 is present at equal levels in interphase and mitotic cells, and anti-GST Western blots of the beads used for immunoprecipitation demonstrated that equal amounts of GST-Gin4 were precipitated from interphase and mitotic cells. The cell cycle arrest point is indicated above each panel (I, interphase; M, mitosis). Note that the gels used for these Western blots were not run long enough to give full resolution of Gin4 isoforms and therefore appear somewhat different from the Western blots shown in Figure 1.

Figure 3

Figure 3

Purification of a Gin4 multiprotein complex from cells arrested in mitosis. (A) Yeast cells carrying an HA-tagged_GIN4_ were arrested in mitosis, and a crude extract from the cells was incubated with anti-HA beads or anti-GST control beads. After washing, both columns were eluted with an excess of HA dipeptide. Samples from each step of the purification procedure were loaded onto a 10% SDS-polyacrylamide gel, which was stained with Coomassie blue. The lanes marked “LSS” and “HSS” are the low- and high-speed supernatants, respectively, and the lane marked “flowthrough” represents the unbound proteins from the anti-HA immunoaffinity column. Asterisk denotes the antibody heavy chain. The arrowheads denote two background proteins that we consistently see in our purifications. Mass spectrometry analysis identifies these as Ssa1 and Ssa2. (B) The same fractions shown in A were loaded onto a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Fractions were probed with anti-Gin4-HA, anti-Shs1, anti-Cdc11, and anti-Nap1 antibodies.

Figure 4

Figure 4

Gin4 associates with Cdc11 in a mitosis-specific manner. (A) Log phase cells were synchronized in G1 by treatment with α factor, and samples were taken every 15 min after release from the arrest. Gin4 was immunoprecipitated (IP) from each sample, and the coprecipitation of Cdc11 and Nap1 was assayed by Western blotting. In addition, the levels of Nap1, Cdc11, and Clb2 present in the crude extracts were assayed by Western blotting. Previous work has shown that levels of Gin4 remain constant during the cell cycle (Altman and Kellogg, 1997). (B) Wild-type and_cdc28-4_ cells were grown to log phase at room temperature and then shifted to 37°C The association of Gin4 with Cdc11 was detected by coprecipitation. (C) Log phase cells containing Cln3 under the control of the Gal promoter were synchronized in G1 by the addition of dextrose-containing media and then released from the arrest. Samples were taken every 30 min and the coprecipitation of Cdc11 and Nap1 was assayed as before.

Figure 5

Figure 5

Mitotic Cdc28 kinase activity is required for Gin4 hyperphosphorylation and complex assembly. (A) Cells containing the cdc28-as1 allele were arrested in mitosis and then treated with 1NM-PP1 or mock treated with DMSO for 15 min. The behavior of the Gin4 protein was assayed by Western blotting. (B) Gin4 was immunoprecipitated (IP) from the indicated samples and coprecipitation of Nap1, Cdc11, and Shs1 was monitored by Western blotting. (C) Western blotting of the same samples shown in A revealed that Clb2 levels remain high in cells treated with 1NM-PP1.

Figure 6

Figure 6

In vivo requirements for complex assembly. (A) Gin4 was immunoprecipitated (IP) from the indicated strains, and coprecipitation of Cdc11 and Nap1 was assayed by Western blotting. Western blots of the crude extracts were carried out to verify equal protein levels in the crude extracts. (B) The Gin4-Gin4 interaction was monitored in the indicated strains by immunoprecipitation of GST-Gin4 and assaying for coprecipitation of 3×HA-Gin4. Note that the gels used for these Western blots were not run long enough to give full resolution of Gin4 isoforms and therefore appear somewhat different from the Western blots shown in Figure 1. (C) Gin4 was immunoprecipitated from the indicated strains arrested in either interphase (I) or mitosis (M), and coprecipitation of Shs1 was monitored by Western blotting.

Figure 7

Figure 7

Shs1 is a phosphoprotein that undergoes Gin4-dependent phosphorylation during mitosis. (A) The indicated strains were arrested in interphase with α factor, or in mitosis with nocodazole, and the behavior of Shs1 was monitored by Western blotting with an anti-Shs1 antibody. The behavior of Shs1 in rapidly growing cells was also analyzed. The asterisk denotes a variable background band that we observed in an Shs1 deletion. (B) Shs1 was immunoprecipitated from cells arrested in mitosis with anti-Shs1 antibody, and the precipitated Shs1 was incubated in the presence (+) or absence (−) of λ-phosphatase. After treatment with phosphatase, differently phosphorylated forms of Shs1 were detected by Western blotting.

Figure 8

Figure 8

Shs1 is a member of the core septin complex and is an in vitro substrate for the Gin4 kinase. (A) 3×HA-Shs1, 3×HA-Gin4, and 3×HA-gin4K48A were purified by immunoaffinity chromatography in the presence of 1 M KCl, and the purified proteins were separated on an SDS-polyacrylamide gel. The gel was stained with Coomassie blue. The 3×HA-Gin4 was purified from cells arrested in mitosis to obtain the active hyperphosphorylated form of Gin4. The asterisk denotes the antibody heavy chain. The arrowheads denote two background bands corresponding to chaperone proteins that we consistently see in our purifications. (B) Purified Gin4 and purified septin complexes were incubated together or alone in the presence of [γ32P]ATP, and the reactions were loaded onto a 10% SDS-polyacrylamide gel. (C) After incubating Gin4 and septin complexes with [γ32P]ATP, protein complexes were disrupted by the addition of 1% SDS and incubation at 100°C. Samples were then incubated with either anti-Shs1 or anti-GST control beads. After incubation, samples were loaded onto a 10% SDS-polyacrylamide gel.

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