The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we o... More
The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during α-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a well-established effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.
