Ssp1 CaMKK: A Sensor of Actin Polarization That Controls Mitotic Commitment through Srk1 in Schizosaccharomyces pombe (original) (raw)
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CAK1 promotes meiosis and spore formation in Saccharomyces cerevisiae in a CDC28-independent fashion
2002
CAK1 encodes a protein kinase in Saccharomyces cerevisiae whose sole essential mitotic role is to activate the Cdc28p cyclin-dependent kinase by phosphorylation of threonine-169 in its activation loop. SMK1 encodes a sporulation-specific mitogen-activated protein (MAP) kinase homolog that is required to regulate the postmeiotic events of spore wall assembly. CAK1 was previously identified as a multicopy suppressor of a weakened smk1 mutant and shown to be required for spore wall assembly. Here we show that Smk1p, like other MAP kinases, is phosphorylated in its activation loop and that Smk1p is not activated in a cak1 missense mutant. Strains harboring a hyperactivated allele of CDC28 that is CAK1 independent and that lacks threonine-169 still require CAK1 to activate Smk1p. The data indicate that Cak1p functions upstream of Smk1p by activating a protein kinase other than Cdc28p. We also found that mutants lacking CAK1 are blocked early in meiotic development, as they show substantial delays in premeiotic DNA synthesis and defects in the expression of sporulation-specific genes, including IME1. The early meiotic role of Cak1p, like the postmeiotic role in the Smk1p pathway, is CDC28 independent. The data indicate that Cak1p activates multiple steps in meiotic development through multiple protein kinase targets.
Fission Yeast Mitotic Regulator Dsk1 Is an SR Protein-specific Kinase
Journal of Biological Chemistry, 1998
Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known to bind the in vivo phosphoepitope shared by SR proteins, indicating that the phosphorylation sites resided in the RS domain. Moreover, the fission yeast U2AF65 homolog, Prp2/Mis11 protein, was phosphorylated more efficiently by Dsk1 than by a human SR protein-specific kinase, SRPK1. Thus, these in vitro results suggest that Dsk1 is a fission yeast SR protein-specific kinase, and Prp2/Mis11 is likely an in vivo target for Dsk1. Together with previous genetic data, the studies support the notion that Dsk1 may play a role in coordinating pre-mRNA splicing and the cell division cycle.
Journal of Cell Biology, 2010
The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosis-to-interphase...
A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast
2011
Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2A Cdc55) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2A Cdc55 sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2A Cdc55 and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength.
Journal of Biological Chemistry, 2003
In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.
Journal of Biological Chemistry, 2010
In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G 1 /S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G 2 /M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G 1 /S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.
Molecular Biology of the Cell, 2003
Chromosome segregation, mitotic exit, and cytokinesis are executed in this order during mitosis. Although a scheme coordinating sister chromatid separation and initiation of mitotic exit has been proposed, the mechanism that temporally links the onset of cytokinesis to mitotic exit is not known. Exit from mitosis is regulated by the mitotic exit network (MEN), which includes a GTPase (Tem1) and various kinases (Cdc15, Cdc5, Dbf2, and Dbf20). Here, we show that Dbf2 and Dbf20 functions are necessary for the execution of cytokinesis. Relocalization of these proteins from spindle pole bodies to mother daughter neck seems to be necessary for this role because cdc15-2 mutant cells, though capable of exiting mitosis at semipermissive temperature, are unable to localize Dbf2 (and Dbf20) to the “neck” and fail to undergo cytokinesis. These cells can assemble and constrict the actomyosin ring normally but are incapable of forming a septum, suggesting that MEN components are critical for the ...
Proceedings of the National …, 1998
The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine͞threonine protein kinase, belonging to the myotonic dystrophy kinase͞ cot1͞warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34 cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1͞Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1͞Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.