DOI: 10.12688/wellcomeopenres.27245.1 ISSN: 2398-502X

A chemical-genetic approach for stress-independent activation of the fission yeast stress-activated protein kinase pathway

Kenneth E. Sawin, Ankita Gupta, Tatiana Dudnakova, Beste Bayrak, Adam Kovac, Domenico Modaffari, Ana I. Rodriguez-Rodriguez, Monique L. Scott, Ye Dee Tay
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Background The fission yeast stress-activated protein kinase (SAPK) pathway includes a conserved mitogen-activated protein (MAP) kinase cascade that regulates multiple cellular processes and is activated by several types of external stress. Understanding how Sty1, the MAP kinase in the SAPK pathway, controls these processes is complicated by the fact that different stressors may have stressor-specific effects that are difficult to separate from effects of Sty1 activation itself. Moreover, upon stress, Sty1 activation is usually short-lived. Previously, we developed a fission yeast strain, SISA (“Stress-Independent Sty1 Activation”), in which Sty1 kinase activity can be switched on in a sustained manner in the absence of external stress. This depends on multiple mutations in the SAPK pathway, including an analog-sensitive version of Sty1. When SISA cells are grown in the presence of analog-sensitive kinase inhibitors, Sty1 is inhibited, but when inhibitor is removed, Sty1 becomes hyperactivated. Although SISA is useful, it has several limitations. Methods We constructed and validated a more rationally designed strain, SISA4 , that retains the features of the original SISA strain while overcoming its limitations. Results SISA4 is more genetically stable than SISA , easier to use in genetic crosses, and easier to identify by phenotype or genotyping. We show that analog-sensitive kinase inhibitors 4-Amino-1-tert-butyl-3-(1′-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1) and 4-Amino-1-tert-butyl-3-(3-bromobenzyl)pyrazolo[3,4-d]pyrimidine (3-BrB-PP1) are equally potent for inhibiting analog-sensitive Sty1 in vivo , and we determine optimal inhibitor concentrations for converting SISA4 cells from a Sty1-inhibited state to a Sty1-hyperactivated state. We also find that both 1-NM-PP1 and 3-BrB-PP1 have measurable off-target effects in wild-type cells, although these effects are modest and generally do not affect interpretation of experiments. Finally, using SISA4 , we show that the Sty1-activated transcription factor Atf1 plays an unexpected role in maintaining cell-polarity disruption after Sty1 hyperactivation. Conclusions SISA4 provides new insights into investigating how SAPK pathway activation regulates diverse cellular processes.

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