The Conserved NDR Kinase Orb6 Controls Polarized Cell Growth by Spatial Regulation of the Small GTPase Cdc42

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33101-1015, USA.
Current biology: CB (Impact Factor: 9.57). 08/2009; 19(15):1314-9. DOI: 10.1016/j.cub.2009.06.057
Source: PubMed


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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    • "Thus Orb6 kinase likely regulates at least one more Cdc42 regulator in addition to Gef1. The Cdc42 GAP Rga4 is a likely candidate, as it physically (two-hybrid screen) and functionally interacts with Orb6 kinase (Das et al., 2007, 2009). Orb6 kinase possibly regulates both Gef1 and Rga4 to modulate Cdc42 activation, thus controlling cell dimensions and growth symmetry . "
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    ABSTRACT: Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anti-correlated at the two cell tips in fission yeast. Anti-correlation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 Guanine Exchange Factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24-Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips, and reduces the anti-correlation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase Activating Protein (GAP) Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and to promote cell polarization and cell shape emergence. © 2015 by The American Society for Cell Biology.
    Preview · Article · Aug 2015 · Molecular biology of the cell
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    • "We propose an antagonistic regulation, whereby the NdrC is activated by RasG and inactivated by RasB. The previously described interaction between the Schizosaccharomyces pombe Cdc42, a small GTPase, and Orb6, a member of the LATS/NDR group of kinases [42] suggests the possible generality of this type of mechanism, and it will be important to investigate whether the mammalian LATS kinases are also regulated by small GTPases during cell division. "
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    ABSTRACT: Background Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans. Results In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC. Conclusions Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC-null cells, RasG-null cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG.
    Full-text · Article · Jul 2014 · BMC Cell Biology
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    • "Accordingly, simulations predicted that the OSW should rupture at many sites if growth is diffuse and not restricted to a single cap (Figure 5A). To experimentally test this, we assayed spores of the orb6-25 mutant, a mutant in the NDR kinase orb6p, which shuts off polarity establishment at a downstream level when grown at restrictive temperature (Das et al., 2009). These spores grew in a perfectly isotropic manner, with polarity factors diffusely distributed around the surface, and remained round many hours after the typical timing corresponding to WT outgrowth (Figures 5B and 5C). "
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    ABSTRACT: The morphogenesis of single cells depends on their ability to coordinate surface mechanics and polarity. During germination, spores of many species develop a polar tube that hatches out of a rigid outer spore wall (OSW) in a process termed outgrowth. However, how these awakening cells reorganize to stabilize this first growth axis remains unknown. Here, using quantitative experiments and modeling, we reveal the mechanisms underlying outgrowth in fission yeast. We find that, following an isotropic growth phase during which a single polarity cap wanders around the surface, outgrowth occurs when spores have doubled their volume, concomitantly with the stabilization of the cap and a singular rupture in the OSW. This rupture happens when OSW mechanical stress exceeds a threshold, releases the constraints of the OSW on growth, and stabilizes polarity. Thus, outgrowth exemplifies a self-organizing morphogenetic process in which reinforcements between growth and polarity coordinate mechanics and internal organization.
    Preview · Article · Mar 2014 · Developmental Cell
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