Proteins in the Nutrient-Sensing and DNA Damage Checkpoint Pathways Cooperate to Restrain Mitotic Progression following DNA Damage

Abstract

Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA.

A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging

“…Our data demonstrate that Rad53 and mTOR are both regulated by DR and likely act through parallel mechanisms to influence Los1 localization ( Figure 5 ). This fits with prior data suggesting that Rad53 regulates Los1 in response to DNA damage (Ghavidel et al, 2007), similar to Ntg1 translocation during mtDNA stress (Schroeder and Shadel, 2014), as well as evidence that the nutrient responsive hexokinase Hxk2 and cAMP-dependent protein kinase A (PKA) signaling pathway are important for appropriate regulation of the Rad53 checkpoint following DNA damage (Searle et al, 2011). Both Hxk2 and PKA have been previously shown to act within the DR pathway in yeast, further supporting this link (Kaeberlein et al, 2004; Lin et al, 2000).…”

Cross-Talk between Carbon Metabolism and the DNA Damage Response in S. cerevisiae

“…Protein kinase A (PKA) affects glucose sensing at multiple levels [inhibition of Snf1 (Barrett et al, 2012), Rgt1 (Kim and Johnston, 2006, Roy et al, 2014) and Mth1 (Ma et al, 2014)], and is activated by Mec1 inhibition of the PKA regulatory subunit Bcy1 (Searle et al, 2011). We observed that phosphorylation of Mms21 in response to glucose (but not MMS) is reduced by overexpression of the PDE2 phosphodiesterase ( Figures 1b, c ) and that phosphorylation of Mms21 on a PKA consensus motif ( RxxS, Figure 1e ) is affected by changes in PKA activity.…”

Regulation of pol III transcription by nutrient and stress signaling pathways

“…Phosphorylation of the Tpk isozymes affects substrate specificity and phosphorylation of Bcy1 affects the extent of PKA activity in response to cell stresses from carbon source, heat shock, DNA damage and loss of TORC1 activity [43, 116–118, 189]. In exponentially growing cells Tpk2 and Bcy1 are nuclear while Tpks1 and 3 are distributed throughout the cell.…”

“…This crosstalk between the TOR pathway, Sch9 and the CWI pathway via Slt2 alters the extent of Maf1 phosphorylation: a Slt2 deletion strain caused elevated phospho-Maf1 but did not affect pol III transcription [43,151]. In contrast, the phosphorylation of Bcy1 at other sites appears to have different effects on PKA activity towards other substrates [117,118,188,189]. For example, in response to DNA damage, Mec1-dependent phosphorylation of Bcy1 by Mck1 enables PKA regulation of the DNA damage checkpoint and inhibition of mitotic exit.…”