The Nim1 kinase Gin4 has distinct domains crucial for septin assembly, phospholipid binding and mitotic exit.

In fungi, the Nim1 protein kinases, such as Gin4, are important regulators of multiple cell cycle events, including the G2-M transition, septin assembly, polarized growth and cytokinesis. Compelling evidence has linked some key functions of Gin4 with the large C-terminal non-kinase region which, however, is poorly defined. By systematically dissecting and functionally characterizing the non-kinase region of Gin4 in the human fungal pathogen Candida albicans, we report the identification of three new domains with distinct functions: a lipid-binding domain (LBD), a septin-binding domain (SBD) and a nucleolus-associating domain (NAD). The LBD and SBD are indispensable for the function of Gin4, and they alone could sufficiently restore septin ring assembly in GIN4-null mutants. The NAD localizes to the periphery of the nucleolus and physically associates with Cdc14, the ultimate effector of the mitotic exit network. Gin4 mutants that lack the NAD are defective in spindle orientation and exit mitosis prematurely. Furthermore, we show that Gin4 is a substrate of Cdc14. These findings provide novel insights into the roles and mechanisms of Nim1 kinases in the regulation of some crucial cell cycle events.

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth.

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin-dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28-Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28-Clb2 and the hyphal-specific Cdc28-Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip-to-neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.

CDK regulates septin organization through cell-cycle-dependent phosphorylation of the Nim1-related kinase Gin4.

Cyclin-dependent kinases (CDKs) regulate septin organization in a cell-cycle-dependent manner in yeast. However, the mechanism remains unclear. Here, we show that the Candida albicans CDK Cdc28 phosphorylates the Nim1-related kinase Gin4, a known septin regulator, activating its kinase activity, which in turn phosphorylates the Sep7 septin. Gin4 contains a cluster of CDK phosphorylation sites near the kinase domain. Replacing serine/threonine with alanine in these sites prevents Gin4 activation, weakens its association with Sep7, alters Sep7 dynamics and causes morphological and cytokinetic defects. By contrast, phosphomimetic mutation enhances the kinase activity with only moderate deteriorating effects. We also found that Gin4 has both kinase-independent and -dependent functions, acting during G1 phase and mitosis, respectively, with the former being essential for septin ring assembly. Thus, we have identified a previously unknown signaling pathway linking CDKs and the septins that provides new insights into the mechanisms controlling septin organization and function in coordination with cell-cycle phases.