Cell Cycle-dependent Assembly of a Gin4-Septin Complex

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.

Some assembly required: yeast septins provide the instruction manual

“…In this regard, the apparent molecular weight of recombinant complexes isolated at a moderate salt concentration (0.25 M) is consistent with two copies of each septin in a 1:1:1:1 stoichiometry [10], whereas the amount of Cdc11 in native complexes that are isolated at higher salt concentrations (0.5-1.0 M) is substoichiometric [8,38]. Likewise, in high-salt conditions, the amount of Shs1 recovered in native complexes is substoichiometric [39]. Together, these data indicate a model for the complex that is illustrated in Figure 1b.…”

“…Phosphorylation by two protein kinases, Cla4 [28,[66][67][68] and Gin4 [39,48,69], plays a prominent role in initiating and/or stabilizing filament assembly during collar formation during emergence of the bud. Cla4 is a clear-cut ortholog of mammalian p21-activated protein kinases (PAKs), and the closest mammalian relative of Gin4 is the neuron-specific, AMPK-related, kinase-family member, BRSK1.…”

Shs1 Plays Separable Roles in Septin Organization and Cytokinesis in Saccharomyces cerevisiae

“…To fully understand the phenotypic differences between shs1D cdc10D strain and shs1D cdc11D strain, it is essential to perform analysis of subunit composition of the septin complex produced in these double mutants. Moreover, Mortensen et al (2002) reported that Shs1 interacts with Gin4 kinase independently of Cdc11 in cla4D and nap1D cells. Cdc3 is phosphorylated in vivo, which plays a role in septin ring Figure 7.-Phenotypes of the shs1-100c allele.…”

The pathobiology of the septin gene family

“…Furthermore, this is associated with coordinated regulation of septin interactions [58] and with a role for Cdc42 [59]. A compelling model has the septin ring in the yeast bud neck acting as a scaffold [20] for the regulated association and dissociation of kinases such as Cla4/PAK, Nim 1/Gin4, and Cdc5/Polo [60][61][62][63][64][65][66][67]. These can then spatially and temporally regulate the activation status of Swe1 and link morphogenetic events to M-phase entry.…”