Douglas R. Kellogg

Septins: cytoskeletal polymers or signalling GTPases?

Septins are a family of conserved proteins that have been implicated in a variety of cellular functions involving specialized regions of the cell cortex and changes in cell shape. The biochemistry and localization of septins suggest that they form a novel cytoskeletal system or that they function as scaffolds for the assembly of signalling complexes. This article discusses septin biochemistry and septin-interacting proteins, focusing on the missing link between the structure and biochemical properties of septin proteins, and on how they function at a molecular level in processes such as cytokinesis and yeast budding.

Cdk1-dependent regulation of the mitotic inhibitor Wee1.

The Wee1 kinase phosphorylates and inhibits cyclin-dependent kinase 1 (Cdk1), thereby delaying entry into mitosis until appropriate conditions have been met. An understanding of the mechanisms that regulate Wee1 should provide new insight into how cells make the decision to enter mitosis. We report here that Swe1, the budding-yeast homolog of Wee1, is directly regulated by Cdk1. Phosphorylation of Swe1 by Cdk1 activates Swe1 and is required for formation of a stable Swe1-Cdk1 complex that maintains Cdk1 in the inhibited state. Dephosphorylation of Cdk1 leads to further phosphorylation of Swe1 and release of Cdk1. Thus, Cdk1 both positively and negatively regulates its own inhibitor. Regulation of the Swe1-Cdk1 complex is likely to play a critical role in controlling the transition into mitosis.

Conservation of mechanisms controlling entry into mitosis: budding yeast wee1 delays entry into mitosis and is required for cell size control.

BACKGROUND In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis. RESULTS In contrast to previous studies, we found that budding yeast swe1 Delta cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms. CONCLUSIONS Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast.

The elm1 kinase functions in a mitotic signaling network in budding yeast.

ABSTRACT In budding yeast, the Clb2 mitotic cyclin initiates a signaling network that negatively regulates polar bud growth during mitosis. This signaling network appears to require the function of a Clb2-binding protein called Nap1, the Cdc42 GTPase, and two protein kinases called Gin4 and Cla4. In this study, we demonstrate that the Elm1 kinase also plays a role in the control of bud growth during mitosis. Cells carrying a deletion of the ELM1 gene undergo a prolonged mitotic delay, fail to negatively regulate polar bud growth during mitosis, and show defects in septin organization. In addition, Elm1 is required in vivo for the proper regulation of both the Cla4 and Gin4 kinases and interacts genetically with Cla4, Gin4, and the mitotic cyclins. Previous studies have suggested that Elm1 may function to negatively regulate the Swe1 kinase. To further understand the functional relationship between Elm1 and Swe1, we have characterized the phenotype of Δelm1 Δswe1 cells. We found that Δelm1 Δswe1 cells are inviable at 37°C and that a large proportion of Δelm1Δswe1 cells grown at 30°C contain multiple nuclei, suggesting severe defects in cytokinesis. In addition, we found that Elm1 is required for the normal hyperphosphorylation of Swe1 during mitosis. We propose a model in which the Elm1 kinase functions in a mitotic signaling network that controls events required for normal bud growth and cytokinesis, while the Swe1 kinase functions in a checkpoint pathway that delays nuclear division in response to defects in these events.

Mapping and analysis of phosphorylation sites: a quick guide for cell biologists

A mechanistic understanding of signaling networks requires identification and analysis of phosphorylation sites. Mass spectrometry offers a rapid and highly sensitive approach to mapping phosphorylation sites. However, mass spectrometry has significant limitations that must be considered when planning to carry out phosphorylation-site mapping. Here we provide an overview of key information that should be taken into consideration before beginning phosphorylation-site analysis, as well as a step-by-step guide for carrying out successful experiments.

Cdk1 coordinates cell-surface growth with the cell cycle

The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the Cdc42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin–Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the Cdc42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.

Control of mitotic events by the Cdc42 GTPase, the Clb2 cyclin and a member of the PAK kinase family

BACKGROUND Cyclins and cyclin-dependent kinases induce and coordinate the events of the cell cycle, although the mechanisms by which they do so remain largely unknown. In budding yeast, a pathway used by the Clb2 cyclin to control bud growth during mitosis provides a good model system in which to understand how cyclin-dependent kinases control cell-cycle events. In this pathway, Clb2 initiates a series of events that lead to the mitosis-specific activation of the Gin4 protein kinase. A protein called Nap1 is required in vivo for the activation of Gin4, and is able to bind to both Gin4 and Clb2. We have used a simple genetic screen to identify additional proteins that function in this pathway. RESULTS We have found that the Cdc42 GTPase and a member of the PAK kinase family called Cla4 both function in the pathway used by Clb2 to control bud growth during mitosis. Cdc42 and Cla4 interact genetically with Gin4 and Nap1, and both are required in vivo for the mitosis-specific activation of the Gin4 kinase. Furthermore, Cla4 undergoes a dramatic hyperphosphorylation in response to the combined activity of Nap1, the Clb2-Cdc28 kinase complex, and the GTP-bound form of Cdc42. Evidence is presented which suggests that the hyperphosphorylated form of Cla4 is responsible for relaying the signal to activate Gin4. CONCLUSIONS Previous studies have suggested that cyclin-dependent kinases control the cell cycle by directly phosphorylating proteins involved in specific events, such as nuclear lamins, microtubule-associated proteins and histones. In contrast, our results demonstrate that the Clb2-Cdc28 cyclin-dependent kinase complex controls specific cell-cycle events through a pathway that involves a GTPase and at least two different kinases. This suggests that cyclin-dependent kinases may control many cell-cycle events through GTPase-linked signaling pathways that resemble the intricate signaling pathways known to control many other cellular events.

Regulation of Mih1/Cdc25 by protein phosphatase 2A and casein kinase 1

The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression.

Cdc28-Dependent Regulation of the Cdc5/Polo Kinase

Polo kinase is activated as cells enter mitosis and plays a central role in coordinating diverse mitotic events, yet the mechanisms leading to activation of Polo kinase are poorly understood . Work in Xenopus meiotic cell cycles has suggested that Polo kinase functions in a pathway that helps trigger activation of Cdk1 . However, studies in other organisms have suggested that activation of Polo kinase is dependent upon Cdk1 and therefore occurs downstream of Cdk1 activation . In this study, we have investigated the role of Cdk1 in the activation of budding yeast Polo kinase. The budding yeast homologs of Cdk1 and Polo kinase are referred to as Cdc28 and Cdc5. We show that signaling from Cdc28 is required to maintain Cdc5 activity in vivo. Furthermore, purified Cdc28 associated with the mitotic cyclin Clb2 is sufficient to activate purified Cdc5 in vitro. A single Cdc28 consensus phosphorylation site found at threonine 242 in the activation loop segment of Cdc5 is required for Cdc5 function in vivo and for kinase activity in vitro, whereas four other Cdc28 consensus sites are dispensable. Analysis of Cdc5 phosphorylation by mass spectrometry indicates that threonine 242 is phosphorylated in vivo. These results suggest that Cdc28 activates Cdc5 via phosphorylation of threonine 242.

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

The Wee1 kinase inhibits cyclin-dependent kinase 1 (Cdk1) during early mitosis. A low level of Cdk1 activity must escape Wee1 inhibition to initiate early mitotic events, but the underlying mechanisms have remained unknown. In this paper, we show that a specific form of protein phosphatase 2A opposes activation of Wee1, which allows low-level activation of Cdk1 in early mitosis.