The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Here we describe the characterization of fission yeast Mob2p, a novel protein required for regulating cell polarity and cell cycle control. Deletion of mob2 is lethal and causes cells to become spherical, with depolarized actin and microtubule cytoskeletons. A decrease in Mob2p protein level results in a defect in the activation of bipolar growth. This phenotype is identical to that of mutants defective in the orb6 protein kinase gene, and we find that Mob2p physically interacts with Orb6p. In addition, overexpression of Mob2p, like that of Orb6p, results in a delay in the onset of mitosis. Mob2p localizes to the cell periphery and cytoplasm throughout the cell cycle and to the division site during late anaphase and telophase. Mob2p is unable to localize to the cell middle in mutants defective in actomyosin ring and septum formation. Our results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis.
"Previous reports suggest that activation of Ndr kinases is a multistep process involving binding of Mob proteins and phosphorylation at the conserved sites , , and the conserved phosphorylation sites have been shown to be important for the function of several Ndr kinases , , –. The mechanism of Orb6 activation remains unclear, although it was reported that Orb6 interacts with Mob2 , and Orb6 kinase activity is dependent on the MOR network components Pmo25, Nak1 and Mor2 . In this paper, we have further investigated the relationship of Nak1 and Orb6, and the role of conserved phosphorylation sites in Orb6 function. "
[Show abstract][Hide abstract] 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 · 3.23 Impact Factor
"It will be of interest to determine whether the steady-state level of phosphorylation of these sites in Sid2p is affected in PP2A mutants. In S. pombe, the NDR-family kinase Mob2p–Orb6p (Hou et al. 2003) is a component of the morphology network (MOR), which regulates polar growth in S. pombe. It has been suggested that increased SIN activity is antagonistic to the MOR (Ray et al. 2010), and that SIN activity in mitosis turns off polar growth in preparation for cytokinesis; reviewed by Gupta and McCollum (2011). "
[Show abstract][Hide abstract] ABSTRACT: The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis. Cdc7p is the first kinase in the core SIN; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologs of mammalian phosphotyrosyl phosphatase activator. We have characterized this gene and its ortholog SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of protein phosphatase type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major protein phosphatase type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.
"Moreover, it also regulates localization of the F-actin cable polymerization factor, For3 at the cell tips via spatial control of the Cdc42 GTPase, a central regulator of cell polarity . The MOR signaling pathway includes Orb6 (an NDR kinase) ; its binding partner Mob2  and its upstream activator, Nak1 (a STE-20 like kinase) [11,12]. Pmo25 has been identified as a binding partner of Nak1 and is essential for the activities of both kinases in the pathway . "
[Show abstract][Hide abstract] 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.
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