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- Ste5 is regulated according to cell cycle state with 8 phosphorylation sites near N terminus being phosphorylated at the G1-S transition such that scaffold is no longer able to associate with the membrane and cells are unresponsive to pheromone. Strickfaden et al 2007 DOI 10.1016/j.cell.2006.12.032
- Ste5 binds Ste11 with at most residues 336-586 (by two-hybrid with Ste5 fragments). Choi et al. 1994 PMID 8062390
- Ste5 binds Ste11 with at least residues 463-514 (by two-hybrid with Ste5 point mutants), and residues 57-71 may enhance Ste5 affinity for Ste11. Inouye et al. 1997 PMID 9335587
- Ste5 binding to Ste11 and Ste5 mating function are eliminated by I504T or F514L mutation of Ste5; these mutants can bind Ste4 and Ste7 and Fus3 normally. Inouye et al. 1997 PMID 9335587
- Ste5 binds Ste7 with at most residues beyond 586 to the last amino acid 917 (by two-hybrid with Ste5 fragments). Choi et al. 1994 PMID 8062390
- Ste5 binds Ste7 with at least residues 744-895 (by two-hybrid with Ste5 point mutants). Inouye et al. 1997 PMID 9335587
- Ste5 binding to Ste7 and thus mating function are eliminated by V763A S861P mutation of Ste5; this Ste5 mutant is able to bind Ste4 and Ste11 and Fus3 normally. Ste5 mutant D746G strongly reduces but does not eliminate binding to Ste7, permits normal Ste5 binding to its other partners, yet appears to support wild-type levels of mating--suggesting transient or low levels of binding of Ste7 to Ste5 may be sufficient for normal levels of signaling. Inouye et al. 1997 PMID 9335587
- Ste5 binds Fus3 and Kss1 with at most residues 241-336 (by two-hybrid with Ste5 fragments). Choi et al. 1994 PMID 8062390
- Ste5 binds Ste4 with at most residues beyond 1-214 (by two-hybrid with Ste5 fragment). Ste5 and Ste4 can be co-immunoprecipitated from cell extracts. Whiteway et al. 1995 PMID 7667635
- Ste5 binds to Ste4 in a pheromone-dependent manner and preferentially associates with a phosphorylated form of Ste4. Feng et al. 1998 PMID 9501067
- Ste5 dimerizes/oligomerizes with residues 138-239 and 335-586. Yablonski et al. 1996 PMID 8943027
- RING-H2 domain (aka LIM domain, residues 177-229) is implicated in dimerization/oligomerization and in binding to Ste4. Inouye et al. 1997 PMID 9311911
- The RING-H2 domain may inhibit signaling through the scaffold, and cease inhibition upon Ste4 binding.
- Binding of Ste4 to Ste5 may relieve inhibitory function of RING-H2 domain and may promote dimerization of Ste5 (dimerization experiment was very indirect, interallelic complementation).
- RING-H2 domains include -cys-X2-cys-X12-17-cys-X-his-X2-his-X2-cys-X8-39-cys-X2-cys- and coordinate 2 Zn2+ atoms.
- C177S and C177A C180A mutants are thought to disrupt coordination of zinc and were found unable to support mating, pheromone dependent transcription, or growth arrest.
- C177A C180A mutant can co-immunoprecipitate (co-IP) Ste11, Ste7, and Fus3 just like wild-type Ste5 can, but this mutant is unable to co-IP as much Ste4 as wild-type Ste5.
- C177A C180A Ste5 mutant cannot complement a mutant Ste5 that is specifically defective in binding Ste11 or Ste7 (despite the ability of these latter two to exhibit allelic complementation), which may or may not suggest that C177A C180A cannot dimerize with Ste5 having an intact RING domain.
- Fusion of GST (which can homodimerize) in place of the last 4 amino acids of Ste5 can restore full or nearly full functionality to C177A C180A mutant, in mating and transcription assays, yet Ste5(C177A C180A)-GST still cannot bind Ste4 by co-IP.
- Ste5(C177A C180A)-GST can support (somewhat reduced) mating in a ste4Δ ste5Δ strain, although Ste5-GST cannot (this latter chimera requires Ste4 for function).
- RING-H2 domain (aka LIM domain, residues 177-229) is implicated in binding to Ste4. Feng et al. 1998 PMID 9501067
- Ste5(C180A) is impaired in binding to Ste4, but can oligomerize; bind Ste11, Ste7, and Fus3; facilitate basal activation of Ste11 and relay the Ste11 signal to the MAP kinases.
- Ste5(C180A) cannot activate Ste11 in response to pheromone
- The inability of Ste5(C180A) to bind Ste4, and the ability of Ste5(C180A) to oligomerize may be somewhat debatable to to variable basal signal in two-hybrid studies.
- Oligomerization was also measured by co-IP of Myc tagged Ste5 (WT or mutant) with GST tagged Ste5 (WT or mutant). These experiments could be confounded because GST was always used for the pulldowns, and GST causes near complete homo-oligomerization of WT Ste5.
- Ste5 binds Bem1. Bem1 is needed for efficient signal transduction (transcriptional activation, polarized growth, and G1 arrest). Lyons et al. 1996 PMID 8754808. Leeuw et al. 1995 PMID 7502048.
- Computational analysis reveals that there is an optimal MAPK scaffold protein concentration to achieve maximal signaling through the cascade. Below this concentration, there is too little scaffold to make efficient use of the amount of kinases, and above which the scaffold begins to titrate the kinases on to separate scaffolds. Levchenko et al. 2000 PMID 10823939
- In the absence of pheromone, Ste5 (when overexpressed) is distributed thoughout the nucleus and cytoplasm, with slightly higher levels in the nucleus. Upon treatment with pheromone, Ste5 localizes to the membrane and especially the shmoo tip. Pryciak and Huntress. 1998 PMID 9732267
- Overexpression of Ste5 under galactose control activates the pheromone response pathway. This activation is dependent on Ste4 and Ste18 and partially dependent on Ste20. Akada et al. 1996 PMID 8722766
- Within 5 minutes of pheromone treatment, the Ste5 that is localized to the membrane is concentrated at the site of the incipient shmoo tip. Mahanty et al. 1999 PMID 10481914
- The signaling defective Ste5(Δ49-66) mutant (residues 49-66 constitute a putative NLS) is defective for nuclear localization and membrane association. This mutant interact efficeintly with Ste4, Ste11, Ste7 and Fus3, and restores mating when coexpressed with a constitutive Ste11 allele that requires a functional Ste5 scaffold. Mahanty et al. 1999 PMID 10481914
- Ste5 mutant T52M is partially active in absence of pheromone, and its activity is partly dependent and partly independent of Ste4. Hasson et al. 1994 PMID 8289786
- Two constitutive Ste5 alleles (resulting in pathway activation in ste5Δ ste4Δ cells) are point mutations (P44L and S770K) near an N-terminal basic segment (44-67) and a C-terminal acidic segment (770-785). Sette et al. 2000 PMID 11071925
- These mutations were both found to increase association between C and N terminal fragments (by coimmunoprecipitation).
- These mutations did not appear to alter (increase or decrease) Ste5 homo-dimerization/oligomerization (by coimmunoprecipitation).
- It is suspected that since the Ste7 binding site is near the C-terminus, and the Fus3 binding site is near the N-terminus, the constitutive phenotype may be a result of intra- or inter- molecular interactions between the N and C termini of Ste5 aligning bound Ste7 and Fus3 to increase phosphorylation of Fus3 (see figure on right).
- Constitutive Ste5 alleles do not localize to shmoo tip in ste5Δ ste4Δ. A shmoo still forms, suggesting that proteins other than Ste4 are able to recuit the relevant molecules to a single site to produce morphological changes. Sette et al. 2000 PMID 11071925
- Ste5 helps to insulate signals sent through Ste11 for mating, HOG and filamentation pathways. Ste11-Ste5 fusion is competent for mating response, but not for HOG of filamentation response. Harris et al. 2001 PMID 11728304
- Ste5 helps to insulate Ste7 from receiving signals sent through Raf (mammalian) and Bck1 (yeast cell integrity pathway) kinases. Yashar et al. 1995 PMID 8524219
- In the absence of pheromone, Ste5-GFP (overexpressed off the Gal promoter) is mainly found in the nucleus, with significant cytoplasmic localization as well. After 2 hours of pheromone exposure, Ste5-GFP is localized to the shmoo tip. Nuclear localization is somewhat decreased, and cytoplasmic localization is largely unchanged. van Drogen et al. 2001 PMID 11781566
- FRAP (fluorescence return after photobleaching) studies of Ste5-GFP (expressed off the Gal promoter) show that it has a half time of re-entry into the nucleus of 2.0s (+/-0.1s) (half time of recovery to constant nuclear fluorescence). van Drogen et al. 2001 PMID 11781566
- RING domains have been implicated in the formation of supramolecular assemblies in cells. Kentsis et al. 2002 PMID 12438698
- Ste5(Δ474-487) and Ste5(L482/485A) exhibit defects in nuclear localization, membrane localization in response to pheromone, and Ste11 binding (co-IP). Wang and Elion. 2003 PMID 12808050
- These mutants exhibit increased homo-oligomerization, normal hetero-oligomerization with WT Ste5, and almost no detectable hetero-oligomerization with Ste5(ΔRING) (Δ177-229) (co-IP). This suggests that these mutations make the RING-H2 domain more readily accessible for oligomerization.
- Despite the fact that these mutations decrease membrane localization upon pheromone treatment, they associate more with Ste4 in the absence of pheromone than WT Ste5 does (co-IP).
- These mutation also cause increased association with Ste7 in the absence of pheromone (co-IP).
- Ste5(Δ474-487) and Ste5(L482/485A) may experience increased nuclear export due to their increased rate of oligomerization. Wang and Elion. 2003 PMID 12808050
- Deletion of Msn5 (nuclear exportin) results in increased nuclear accumulation. The authors conlcude that this suggests that these mutants have increased nuclear export. Decreased nuclear import could also explain these results.
- Co-expression of WT Ste5 with Ste5(Δ474-487) or Ste5(L482/485A) (which hetero-dimerize weakly with WT Ste5) somewhat increases the nuclear localization of these mutants. The authors use this as evidence towards their conclusion that these mutants experience increased nuclear export (presumably through WT Ste5 disrupting the mutant homo-dimers to decrease the number of dimers). It seems unclear how this supports their conclusion.
- Co-expression of Ste5-Myc9 with a Ste5 mutant that has been tagged with a nuclear localization signal (TAgNLS-Ste5) increases the fraction of Ste5-Myc9 localized to the nucleus. Upon exposure to pheromone, co-expression of Ste5-Myc9 and TAgNLS-Ste5 also increases the fraction of Ste5-Myc9 localized to the plasma membrane. Wang and Elion. 2003 PMID 12808050
- The authors conclude that the TAgNLS-Ste5-mediate enrichment of Ste5-Myc9 at the membrane in response to pheromone is greater than the nuclear enrichment in the absence of pheromone (though their number don't support this - see table 2 rows 8, 10 an 12).
- From this the authors conlcude that the size of the nuclear pool of Ste5 is a limiting factor in the amount of Ste5 that is localized to the membrane in response to pheromone. A more likely conclusion is that co-expression of two Ste5 mutants results in a higher overall Ste5 concentration, leading to more dimer. These dimers are more succeptible to binding Ste4, and thus membrane recruitment.
- Ste5 levels increase after pheromone exposure (judged by immunoblotting Ste5-Myc9). Ste5 mRNA levels were unaffected by pheromone exposure, mutations in Fus3 and Kss1, and mutations in Ste12, suggesting that the increase in Ste5 levels is regulated at a post-trasciptional level. Flotho et al. 2004 PMID 15322134
- Ste5-Myc9 is somewhat phosphorlyated in the absence of pheromone, and this phosphorylation appears to be Cdc28 depdendent, and not dependent on Ste11, Ste7, Fus3 or Kss1. Flotho et al. 2004 PMID 15322134
- Phosphorylation was detected by gel-shift assay. Shift was abolished when Ste5-Myc9 was pretreated with a non-specific phosphatase.
- Ste5 basal phosphorylation is unaffected by deletion of Ste20, Ste11, Ste7 and Fus3/Kss1.
- Ste5 basal phosphorylation was drastically decreased when Cdc28-4 (a temperature-sensitive Cdc28 allele) cells were grown at the restrictive temperature.
- Since this phosphorylation is Cdc28 dependent, it might be cell cycle dependent. The authors did not investigate this.
- Ste5 is phosphorylated in response to pheromone. Active Fus3 and Kss1 appear to be the main contributors to this phosphorylation. Flotho et al. 2004 PMID 15322134
- Ste5 mutants that fail to recruit to the membrane (TAgNLS(K128T)-Ste5 which is predominantly nuclear, and Ste5-C180A which fails to dimerize and bind Ste4) are not phosphorylated in response to pheromone.
- Activation of the pathway via a constitutive Ste11 allele (Ste11-4) fails to result in phosphorylation of Ste5 in the absence of pheromone, suggesting that membrane recruitment (or Ste4-binding) is required for pheromone-dependent Ste5 phosphorylation.
- Ste5-Myc9 coimmunoprecipitates with GFP-Cdc24. Wang et al. 2005 PMID 15657049
- Ste5 residues 37-76 contain an overlapping nuclear localization signal (NLS) and a plasma membrane binding domain (PM) (a putative amphipathic α helix). The authors call this domain the PM/NLS domain. Winters et al. 2005 PMID 16209942
- Ste5’s PM/NLS domain binds acidic phospholipids PIP2 and PI4P. Winters et al. 2005 PMID 16209942
- GST-Ste5(37-76) bound to liposomes containing phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI2).
- Mutation of Stt4 (a PI 4-kinase that is responsible for production of PI4P at the plasma membrane) nearly eliminated Ste5(1-214)’s constitutive membrane association.
- Ste5 mutations that increase the PM/NLS domain’s affinity for the plasma membrane lead to a hyperactive phenotype. Winters et al. 2005 PMID 16209942
- Ste5(E756G) appears to route pheromone signal away from Fus3 and towards Kss1. Schwartz and Madhani. 2006 PMID 1646342
- Fluorescently-tagged Ste5, expressed at wild-type levels, is distributed evenly throughout the nucleus and cytoplasm in the absence and presence of pheromone. Maeder et al. 2007 PMID 17952059
<modelMoleculeType>Ste5(Ste5_site, Ste4_site, Ste11_site, Ste7_site, MAPK_site)</modelMoleculeType>
<modelSeedSpecies>Ste5(Ste5_site, Ste4_site, Ste11_site, Ste7_site, MAPK_site) Ste5_tot_conc</modelSeedSpecies>