The Conserved NDR Kinase Orb6 Controls Polarized Cell Growth by Spatial Regulation of the Small GTPase Cdc42
ABSTRACT The conserved NDR kinase regulates cell morphogenesis and polarized cell growth in different eukaryotic cells ranging from yeast to neurons. Although studies have unraveled the mechanism of regulation of NDR kinase activity, the mechanism of morphology control by NDR and the effectors that mediate NDR function are unknown. Via a chemical genetic approach, we show that the fission yeast NDR homolog, Orb6 kinase, maintains polarized cell growth at the cell tips by spatially regulating the localization of Cdc42 GTPase, a key morphology regulator. Loss of Orb6 kinase activity leads to the recruitment of Cdc42 GTPase and the Cdc42-dependent formin For3, normally found only at the cell tips, to the cell sides. Furthermore, we show that loss of Orb6 kinase activity leads to ectopic lateral localization of the Cdc42 guanine nucleotide exchange factor (GEF) Gef1, but not of the other Cdc42 GEF, Scd1. Consistent with these observations, gef1 deletion suppresses the increased cell diameter phenotype of orb6 mutants. In contrast, the microtubule cytoskeleton and the localization of the microtubule-dependent polarity markers Tea1 and Tea4 are not altered by loss of Orb6 kinase activity. Our findings indicate that the conserved NDR kinase Orb6 regulates cell polarity by spatially restricting the localization and activity of Cdc42 GTPase.
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ABSTRACT: Regulation of cytoskeletal remodeling is essential for cell cycle transitions. In fission yeast two NDR kinase signaling cascades, MOR and SIN, regulate the actin cytoskeleton to promote polarized growth during interphase and cytokinesis respectively. Our understanding of how these signaling pathways are coordinated to assist transition between the two cell-cycle stages is limited. Here, we review work from our laboratory, which reveals that cross talk between the SIN and MOR pathways is required for inhibition of interphase polarity programs during cytokinesis. Given the conservation of NDR kinase signaling pathways, our results may define general mechanisms by which these pathways are coordinated in higher organisms.Cell Division 11/2011; 6:19. DOI:10.1186/1747-1028-6-19
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ABSTRACT: The Ndr-related Orb6 kinase is a key regulator of polarized cell growth in fission yeast, however the mechanism of Orb6 activation is unclear. Activation of other Ndr kinases involves both autophosphorylation and phosphorylation by an upstream kinase. Previous reports suggest that the Nak1 kinase functions upstream from Orb6. Supporting this model, we show that HA-Orb6 overexpression partially restored cell polarity in nak1 ts cells. We also demonstrated by coimmunoprecipitation and in vitro binding assays that Nak1 and Orb6 physically interact, and that the Nak1 C-terminal region is required for Nak1/Orb6 complex formation in vivo. However, results from in vitro kinase assays did not show phosphorylation of recombinant Orb6 by HA-Nak1, suggesting that Orb6 activation may not involve direct phosphorylation by Nak1. To investigate the role of Orb6 phosphorylation and activity, we substituted Ala at the ATP-binding and conserved phosphorylation sites. Overexpression of kinase-dead HA-Orb6(K122A) in wild-type cells resulted in a loss of cell polarity, suggesting that it has a dominant-negative effect, and it failed to rescue the polarity defect of nak1 or orb6 ts mutants. Recombinant GST-Orb6(S291A) did not autophosphorylate in vitro suggesting that Ser291 is the primary autophosphorylation site. HA-Orb6(S291A) overexpression only partially rescued the orb6 polarity defect and failed to rescue the nak1 defect, suggesting that autophosphorylation is important for Orb6 function. GST-Orb6(T456A) autophosphorylated in vitro, indicating that the conserved phosphorylation site at Thr456 is not essential for kinase activity. However, HA-Orb6(T456A) overexpression had similar effects as overexpressing kinase-dead HA-Orb6(K122A), suggesting that Thr456 is essential for Orb6 function in vivo. Also, we found that both phosphorylation site mutations impaired the ability of Myc-Nak1 to coimmunoprecipitate with HA-Orb6. Together, our results suggest a model whereby autophosphorylation of Ser291 and phosphorylation of Thr456 by an upstream kinase promote Nak1/Orb6 complex formation and Orb6 activation.PLoS ONE 05/2012; 7(5):e37221. DOI:10.1371/journal.pone.0037221