Abstract #20

The identification of the GTPase activating protein Rga2 as a target of the cyclin-dependent kinase Pho85. Richelle Sopko^1,3, Dongqing Huang¹, Brenda Andrews^1,2,3. 1) Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada; 2) Banting and Best Department of Medical Research, University of Toronto, Toronto ON, Canada; 3) Department of Medical Genetics and Microbiology, University of Toronto, Toronto ON, Canada.
   Cell polarization requires regulated cycling of Cdc42 between GTP and GDP bound states. This cycling is perpetuated by the antagonistic activity of two types of factors, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In a systematic synthetic dosage lethality (SDL) screen using a yeast overexpression array, we found that overexpression of the gene encoding Rga2, a Cdc42 GAP, causes lethality in a strain deleted for the Pho85 cyclin-dependent kinase (CDK). We reason that since many known Pho85 substrates are negatively regulated by phosphorylation, this lethality may reflect an accumulation of unmodified substrate in the absence of Pho85 kinase. We have gathered biochemical and genetic evidence to verify Rga2 is a bona fide Pho85 substrate. Pcl-Pho85 kinase complexes phosphorylate Rga2 in vitro and Rga2 phosphoforms peak during late G1 phase, concomitant with Pcl1-Pho85 and Pcl2-Pho85 kinase activity. The relevant G1-specific Pho85 regulatory cyclins co-localize with Rga2 at the incipient bud site. Toxicity due to overexpression of RGA2 is specific to these regulatory mutants and both Pcl1 and Pcl2 interact physically with Rga2. Mutation of 8 of 18 potential Pho85 phosphorylation sites in RGA2 results in toxicity in wild-type cells when this construct is overexpressed. This sensitive genetic assay allows us to prioritize potential phosphorylation sites in terms of their likely biological importance. Inhibition of Rga2 may also involve the kinase activity of Cln-Cdc28 complexes as cln1cln2 mutants, like pho85 mutants, are incapable of coping with overexpression of RGA2. Cln-Cdc28 kinases can also phosphorylate Rga2 in vitro, and interestingly several modified sites are unique to this CDK. The inhibition of GAPs by CDK phosphorylation in yeast may function as a mechanism to promote G1 phase progression.

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