NDR/LATS-family protein kinase genes are indispensable for embryogenesis in Arabidopsis.
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ABSTRACT:
SUBMITTER: Yoon HS
PROVIDER: S-EPMC8409290 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1's phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine -5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2's partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2's interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase.
| S-EPMC2517623 | biostudies-literature
Project description:The plastid is an organelle vital to all photosynthetic and some non-photosynthetic eukaryotes. In the model plant Arabidopsis thaliana, a number of nuclear genes encoding plastid proteins have been found to be necessary for embryo development. However, the exact roles of plastids in this process remain largely unknown. Here we use publicly available datasets to obtain insights into the relevance of plastid activities to A. thaliana embryogenesis. By searching the SeedGenes database (http://www.seedgenes.org) and recent literature, we found that, of the 339 non-redundant genes required for proper embryo formation, 108 genes likely encode plastid-targeted proteins. Nineteen of these genes are necessary for development of preglobular embryos and/or their conversion to globular embryos, of which 13 genes encode proteins involved in non-photosynthetic metabolism. By contrast, among 38 genes which are dispensable for globular embryo formation but necessary for further development, only one codes for a protein involved in metabolism. Products of 21 of the 38 genes play roles in plastid gene expression and maintenance. Examination of RNA profiles of embryos at distinct growth stages obtained in laser-capture microdissection coupled with DNA microarray experiments revealed that most of the identified genes are expressed throughout embryo morphogenesis and maturation. These findings suggest that metabolic activities are required at preglobular and throughout all stages of embryo development, whereas plastid gene expression becomes necessary during and/or after the globular stage to sustain various activities of the organelle including photosynthetic electron transport.
| S-EPMC2944999 | biostudies-literature
Project description:Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/LatsNTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.
| S-EPMC8341033 | biostudies-literature
Project description:NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.
| S-EPMC6367570 | biostudies-literature
Project description:Saccharomyces cerevisiae Cbk1 is a LATS/Ndr protein kinase and a downstream component of the regulation of Ace2 and morphogenesis (RAM) signaling network. Cbk1 and the RAM network are required for cellular morphogenesis, cell separation, and maintenance of cell integrity. Here, we examine the phenotypes of conditional cbk1 mutants to determine the essential function of Cbk1. Cbk1 inhibition severely disrupts growth and protein secretion, and triggers the Swe1-dependent morphogenesis checkpoint. Cbk1 inhibition also delays the polarity establishment of the exocytosis regulators Rab-GTPase Sec4 and its exchange factor Sec2, but it does not interfere with actin polarity establishment. Cbk1 binds to and phosphorylates Sec2, suggesting that it regulates Sec4-dependent exocytosis. Intriguingly, Cbk1 inhibition causes a >30% decrease in post-Golgi vesicle accumulation in late secretion mutants, indicating that Cbk1 also functions upstream of Sec2-Sec4, perhaps at the level of the Golgi. In agreement, conditional cbk1 mutants mislocalize the cis-Golgi mannosyltransferase Och1, are hypersensitive to the aminoglycoside hygromycin B, and exhibit diminished invertase and Sim1 glycosylation. Significantly, the conditional lethality and hygromycin B sensitivity of cbk1 mutants are suppressed by moderate overexpression of several Golgi mannosyltransferases. These data suggest that an important function for Cbk1 and the RAM signaling network is to regulate growth and secretion via Golgi and Sec2/Sec4-dependent processes.
| S-EPMC2592669 | biostudies-literature
Project description:Adaptation to the nutritional environment is critical for all cells. RAS GTPase is a highly conserved GTP-binding protein with crucial functions for cell growth and differentiation in response to environmental conditions. Here, we describe a novel mechanism connecting RAS GTPase to nutrient availability in fission yeast. We report that the conserved NDR/LATS kinase Orb6 responds to nutritional cues and regulates Ras1 GTPase activity. Orb6 increases the protein levels of an Ras1 GTPase activator, the guanine nucleotide exchange factor Efc25, by phosphorylating Sts5, a protein bound to efc25 mRNA. By manipulating the extent of Orb6-mediated Sts5 assembly into RNP granules, we can modulate Efc25 protein levels, Ras1 GTPase activity, and, as a result, cell growth and cell survival. Thus, we conclude that the Orb6-Sts5-Ras1 regulatory axis plays a crucial role in promoting cell adaptation, balancing the opposing demands of promoting cell growth and extending chronological lifespan.
| S-EPMC6740195 | biostudies-literature
Project description:MOB proteins are integral components of signaling pathways controlling important cellular processes, such as mitotic exit, centrosome duplication, apoptosis, and cell proliferation in eukaryotes. The human MOB protein family consists of six distinct members (human MOB1A [hMOB1A], -1B, -2, -3A, -3B, and -3C), with hMOB1A/B the best studied due to their putative tumor-suppressive functions through the regulation of NDR/LATS kinases. The roles of the other MOB proteins are less well defined. Accordingly, we characterized all six human MOB proteins in the context of NDR/LATS binding and their abilities to activate NDR/LATS kinases. hMOB3A/B/C proteins neither bind nor activate any of the four human NDR/LATS kinases. We found that both hMOB2 and hMOB1A bound to the N-terminal region of NDR1. However, our data suggest that the binding modes differ significantly. Our work revealed that hMOB2 competes with hMOB1A for NDR binding. hMOB2, in contrast to hMOB1A/B, is bound to unphosphorylated NDR. Moreover, RNA interference (RNAi) depletion of hMOB2 resulted in increased NDR kinase activity. Consistent with these findings, hMOB2 overexpression interfered with the functional roles of NDR in death receptor signaling and centrosome overduplication. In summary, our data indicate that hMOB2 is a negative regulator of human NDR kinases in biochemical and biological settings.
| S-EPMC2937529 | biostudies-literature
Project description:Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.
| S-EPMC4428629 | biostudies-literature
Project description:The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.
| S-EPMC6941128 | biostudies-literature
Project description:The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutant AtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1(T463A)-CFP and AtSERK1(3T-->A)-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in the AtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of the AtSERK1 endocytosis mechanism.
| S-EPMC186368 | biostudies-literature