[N1] This research was funded by (1) a Research Scholar grant to Eric L. Weiss from the American Cancer Society; Brian J. Yeh is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation.

[N2] In addition to Eric L. Weiss, other authors of the paper are Emily Mazanka (lead author), Brian J. Yeh and Patrick Charoenpong, from Northwestern University; and Jes Alexander, Drew M. Lowery and Michael Yaffee, from the Massachusetts Institute of Technology (MIT).

[N3] Abbreviations. CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; GMC, ganglion mother cells; LMB, leptomycin B; NES, nuclear export sequence; RAM, Regulation of Ace2 and Morphogenesis; RT-PCR, real-time polymerase chain reaction.

The NDR/LATS Family Kinase Cbk1 Directly Controls Transcriptional Asymmetry. Emily Mazanka, Jess Alexander, Brian J. Yeh, Patrick Charoenpong, Drew M. Lowery, Michael Yaffe, Eric L. Weiss. PLoS Biology 6(8) e203. doi: 10.1371 / journal.pbio.0060203  [ Download PDF ]

Abstract

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.

Author Summary

Cells can differentiate by segregating molecules that direct expression of specific sets of genes to one of the two cells produced by division. This generally occurs by direct mechanical movement or asymmetric anchoring of these molecules, which act after division to influence gene expression. In this study, we define a different mechanism by which the budding yeast transcription regulator Ace2 is asymmetrically partitioned. We show that Ace2 moves from uniform distribution to strong accumulation in the daughter nucleus while mother and daughter cells are still connected, and that the enzyme Cbk1 directly controls this segregation by attaching phosphate to specific sites on Ace2. We also demonstrate that Cbk1 is restricted to the daughter cell. Using both biochemical and live-cell experiments, we show that the Cbk1-mediated modifications activate Ace2 and block its interaction with nuclear export machinery, trapping it in the daughter cell nucleus. In addition to demonstrating Cbk1's remarkable biochemical similarity to related enzymes in multicellular organisms, our analysis shows that a uniformly distributed regulator of gene expression can be made asymmetrically active in connected cells through the direct action of a localized modifying enzyme.