In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5.
The Cdc5 protein of budding yeast is a polo-like kinase that has multiple roles in mitosis including control of the mitotic exit network (MEN). MEN activity brings about loss of mitotic kinase activity so that the mitotic spindle is disassembled and cytokinesis can proceed. Activity of the MEN is regulated by a small GTPase, Tem1, which in turn is controlled by a two-component GTPase-activating protein (GAP) formed by Bfa1 and Bub2. Bfa1 has been identified as a regulatory target of Cdc5 but there are conflicting deductions from indirect in vivo assays as to whether phosphorylation inhibits or stimulates Bfa1 activity. To resolve this question, we have used direct in vitro assays to observe the effects of phosphorylation on Bfa1 activity. We show that when Bfa1 is phosphorylated by Cdc5, its GAP activity with Bub2 is inhibited although its ability to interact with Tem1 is unaffected. Thus, in vivo inactivation of Bfa1-Bub2 by Cdc5 would have a positive regulatory effect by increasing levels of Tem1-GTP so stimulating exit from mitosis.
Geymonat
Marco
M
Divisions of Yeast Genetics and Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, Great Britain.
Spanos
Ad
A
Walker
Philip A
PA
Johnston
Leland H
LH
Sedgwick
Steven G
SG
D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D020559:Monomeric GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000494:pharmacology* / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins / Q000187:drug effects / Q000378:metabolism*; D004718:Saccharomycetales / Q000737:chemistry*
C119117:BFA1 protein, S cerevisiae; C070351:BUB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D029701:Saccharomyces cerevisiae Proteins; C089965:TEM1 protein, S cerevisiae; D006160:Guanosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; D020559:Monomeric GTP-Binding Proteins