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Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p

Nature Cell Biology volume 2, pages 677–685 (2000)Cite this article

Abstract

The p21-activated kinases (PAKs) are effectors for the Rho-family GTPase Cdc42p. Here we define the in vivo function of the kinase activity of the budding yeast PAK Cla4p, using cla4 alleles that are specifically inhibited by a cell-permeable compound that does not inhibit the wild-type kinase. CLA4 kinase inhibition in cells lacking the partially redundant PAK Ste20p causes reversible SWE1-dependent cell-cycle arrest and gives rise to narrow, highly elongated buds in which both actin and septin are tightly polarized to bud tips. Inhibition of Cla4p does not prevent polarization of F-actin, and cytokinesis is blocked only in cells that have not formed a bud before inhibitor treatment; cell polarization and bud emergence are not affected by Cla4p inhibition. Although localization of septin to bud necks is restored in swe1Δ cells, cytokinesis remains defective. Inhibition of Cla4p activity in swe1Δ cells causes a delay of bud emergence after cell polarization, indicating that this checkpoint may mediate an adaptive response that is capable of promoting budding when Cla4p function is reduced. Our data indicate that CLA4 PAK activity is required at an early stage of budding, after actin polarization and coincident with formation of the septin ring, for early bud morphogenesis and assembly of a cytokinesis site.

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Figure 1: cla4 alleles that confer sensitivity to 1NM-PP1.
Figure 2: Phenotypes associated with inhibition of Cla4p.
Figure 3: Effect of Cla4p inhibition on cytokinesis and bud emergence.
Figure 4: Phenotypic variation among cla4-75 ts degron strains.
Figure 5: Phenotype of 1NM-PP1-treated cla4-as3 swe1Δ cells.
Figure 6: Reversibility of Cla4p inhibition.

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Acknowledgements

We thank H. Tjandra, D. Kellogg, M. Longtine, M. Shulewitz, J. Thorner, F. Cvrcova, S. Holly and K. Blumer for providing essential strains, plasmids, and antibodies, and S. Biggins and J. Li for assistance with elutriation. We also thank J. Ubersax and members of the Drubin and Barnes laboratories, particularly M. J. T. V. Cope, M. Duncan, K. Kozminski and D. Seikhaus, for helpful discussions. E.L.W. is supported by an American Cancer Society postdoctoral fellowship; support for this work was also provided by a grant from the National Institutes of Health (to D.G.D.).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, 94720-3202, California, USA

    Eric L. Weiss & David G. Drubin

  2. Department of Chemistry, Princeton University, Princeton, 08544, New Jersey, USA

    Anthony C. Bishop

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 94143, California, USA

    Kevan M. Shokat

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Correspondence to David G. Drubin.

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Weiss, E., Bishop, A., Shokat, K. et al. Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p. Nat Cell Biol 2, 677–685 (2000). https://doi.org/10.1038/35036300

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