Imported:YPM/Cell cycle regulation of pheromone response

Category:Yeast Pheromone Response Model Back to main model page

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• Cln2 (and to a lesser extent Cln1), together with Cdc28, acts to repress pheromone response in a cell cycle dependent manner. Oehlen and Cross. 1994 PMID 7926787
  • Deletion of CLN1 and CLN2 eliminates cell cycle repression of FUS1 expression.
  • Overexpression of CLN1 or CLN2 represses pheromone-dependent FUS1 expression.
  • Overexpression of CNL2 eliminates pheromone-dependent cell-cycle arrest (with or without Far1).
  • Overexpression of CNL1 only eliminates pheromone-dependent cell-cycle arrest in far1Δ cells.
  • Basal FUS1 transcription is also regulated in a cell cycle dependent manner, and overexpression of CLN2 eliminates

• Cln2 represses mating response at the level of Ste11. Wassmann and Ammerer. 1997 PMID 9148934
  • The activity of Ste7 and Fus3 vary in a cell-cycle dependent manner.
  • Overexpression of Cln2 represses mating response caused by a hyperactive Ste11 allele, but not the mating response caused by a Gpa1 deletion.

• Ste20 is phosphorylated by Cln2-Cdc28 during the S phase of the cell cycle. This phosphorylation does not appear to affect in vitro Ste20 kinase activity using immunoprecipitated Ste20. Wu et al. 1998 PMID 9774429

• Although the cellular localization of Ste20 is regulated in a cell-cycle dependent manner, the activity of immunoprecipitated Ste20 is independent of cell-cycle. Wu et al. 1998 PMID 9774429

• Cln2-Cdc28 represses mating response via Ste5's PM domain. Strickfaden et al. 2007 PMID 17289571
  • Overexpressed Ste5(Q59L) or Ste11-Cpr, which are sufficient for pathway activation but require the Ste5 PM domain, can be repressed by overexpression of Cln2.
  • Overexpressed Ste5-CTM or Ste11ΔN, which are sufficient for pathway activation but do not require the Ste5 PM domain, cannot be repressed by overexpression of Cln2.

• Cln2-Cdc28 regulates mating response by phosphorylating Ste5 at 8 different sites around Ste5's PM domain. Strickfaden et al. 2007 PMID 17289571
  • Ste5 has 8 CDK phosphorylation sites (SP or TP) near its PM domain.
  • Mutation of these 8 phosphorylation sites to alanines (AP or TP) eliminates cell cycle dependent Fus1-lacZ expression and Fus3 activation, and the ability of overexpressed Cln2 to repress mating response and membrane localization of Ste5.
  • The Ste5(Q59L) mutant, which is Gβγ-independent, can also be rendered immune to Cln2 repression via the same 8A mutations, indicating that Cln2 does not act at the level of the Ste5/Gβγ interaction.
  • All single alanine mutants displayed a small amount of resistance to Cln2-mediated signal repression, suggesting that phosphorylation at all 8 sites is required for full Cln2-mediated repression.
  • Double, triple, and quadruple alanine mutants displayed increasing resistance to Cln2-mediated signal repression.
  • Only the 8A mutant (5A, 6A, and 7A were not tested) displayed full resistance to Cln2-mediated signal repression.
  • Purified Cln2-Cdc28 phosphorylates purified Ste5(1-125) (which contains the PM domain and surrounding phosphorylation sites), but only weakly phosphorylates Ste5(1-125)8A.

• Cln2-mediated inhibition of mating signaling is caused by added negative charges of phosphate groups, and acts specifically on the PM domain. Strickfaden et al. 2007 PMID 17289571
  • Mutation of the 8 phosphorylation sites to glutamates causes partial inhibition of mating signal.
  • Mutation of the 8 SP/TP pairs to EE pairs (16E mutant), to mimic the two negative charges per SP/TP pair caused by phosphorylation, represses response to pheromone as much as Cln2 overexpression.
  • The 14E mutant is slightly more sentitive to pheromone than the 16E mutant, or than WT Ste5 with Cln2 overexpression, suggesting that full phosphorylation is necessary for full signal repression.
  • The 16E mutant is expressed at WT levels, and immunoprecipitates with Ste4 as well as WT Ste5 does.
  • The 16E mutant repression is bypassed by other mutants that activate the pathway in the absence of the Ste5 PM domain.

• Far1 is normally required for pheromone-dependent cell cycle arrest because Far1 inhibits Cdc28 activity, and thus prevents Cln2-Cdc28 from phosphorylating and inhibiting Ste5. Strickfaden et al. 2007 PMID 17289571
  • Far1 is not required for pheromone-mediated cell cycle arrest in Ste5(8A) cells.
  • The Ste58A mutant causes significant cell cycle arrest outside of G1. The fraction of cells that arrest outside of G1 is further increased upon deletion of Far1.
  • Thus cell cycle arrest is not mediated by Far1, rather Far1 is required to prevent cell cycle dependent downregulation of mating signaling, which in turn prevents cell cycle arrest.

• A simple model of Ste5 phosphorylation and membrane association suggests that the ability of Ste5 to associate with the membrane could drop sharply over a narrow range of Cln2-Cdc28 activity. Serber and Ferrell. 2007 PMID 17289565
  • Using a previously published estimate of a 10x reduction in binding per phosphorylation event, a simple kinetic model predicts a switch-like drop in membrane-associated Ste5 in response to changes in active CDK concentration, with a Hill coefficient of 4.1.

• Upon chemical inhibition of Cdc28, asynchronous population of cells have a similar response to pheromone as a synchronous population of cell in G1 phase (not treated with the inhibitor). Colman-Lerner et al. 2005 PMID 16170311
  • This suggests that dephosphorylation of Ste55 is rapid.