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TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.

ABSTRACT: The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. We observe that TORC1 alters the phosphorylation pattern of Cbk1 and we identify a residue within Cbk1 activation loop, T574, for which a phosphomimetic substitution makes Cbk1 catalytically inactive and, indeed, reproduces TORC1 control over cell separation. In addition, we identify the exocyst component Sec3 as a key substrate of Cbk1, since Sec3 activates the SNARE complex to promote membrane fusion. TORC1 activity ultimately compromises the interaction between Sec3 and a t-SNARE component. Our data indicate that TORC1 negatively regulates cell separation in budding yeast by participating in Cbk1 phosphorylation, which in turn controls the fusion of secretory vesicles transporting hydrolase at the site of division.

SUBMITTER: Foltman M

PROVIDER: S-EPMC10468069 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.

Foltman Magdalena M Mendez Iván I Bech-Serra Joan J JJ de la Torre Carolina C Brace Jennifer L JL Weiss Eric L EL Lucas María M Queralt Ethel E Sanchez-Diaz Alberto A

PLoS biology 20230830 8

The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. W ...[more]

PMID: 37647291

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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.

Project description:The regulation of Ace2 and morphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans The RAM pathway's two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2, only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae, Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast-phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hypha transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hypha-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hypha-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicansIMPORTANCE The regulation of Ace2 and morphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects of C. albicans pathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis: suppression of the hypha-to-yeast transition. Very few regulators of this transition have been described, and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2.

Dataset Information

TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.

Publications

TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.

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