Novel Role for an HPt Domain in Stabilizing the Phosphorylated State of a Response Regulator Domain

Abstract

Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain. These HPt domains serve an essential role as histidinephosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another. In Saccharomyces cerevisiae, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7. Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours). However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system. Two-component signal transduction systems in prokaryotic and eukaryotic organisms regulate cellular responses to environmental changes (6, 11). In their simplest form, these regulatory pathways involve an autophosphorylating transmembrane histidine kinase and a cytoplasmic response regulator that is phosphorylated on an aspartic acid residue. Modified and expanded versions of two-component regulatory pathways requiring multiple phosphoryl transfer reactions between several phosphodonor and phosphoreceiver domains have also been identified (1,6,25,27). Some of the more complex phosphorelay systems that have been described thus far include a hybrid sensor kinase (that also contains a phosphoaspartate receiver domain), a histidine-containing phosphotransfer (HPt) protein, and one or more response regulator proteins (4,5,7,8,14,29,34). HPt domains serve a dual purpose as a phosphoreceiver and phosphodonor in order to shuttle phosphoryl groups between two or more response regulator domains. It has also been suggested that the presence of HPt domains and use of multistep phosphorelay systems provide for additional points of regulation of signaling pathways (1, 11).In most cases, response regulator proteins are activated upon phosphorylation. Hence, the intrinsic lifetime of the phosphorylated state of a response regulator is an important factor in determining the duration of the cellular response. Phosphorylated half-lives ranging from seconds for CheY and CheB (10, 37) to several hours for OmpR and Spo0F (13, 40) have been observed. In addition to the intrinsic phosphatase activity of the respon...

Osmotic Stress Signaling and Osmoadaptation in Yeasts

“…Subsequently, the phosphate group is transferred to His64 on Ypd1p and further to Asp554 on Ssk1p. This sequence of events has been demonstrated convincingly by analysis of truncated and mutated proteins both in vivo and in vitro (256)(257)(258)476). These studies also demonstrate that in vivo the histidine kinase cannot bypass the Sln1p response regulator.…”

“…Transfer from Sln1p His576 to Asp1144 was reported to be unidirectional, while that from Sln1p Asp1144 to Ypd1p His64 was reversible. The entire reaction is, however, driven towards phosphorylation of Ssk1p, since the equilibrium of the reaction from Ypd1p His64 to Ssk1p Asp554 was on the side of the latter (256)(257)(258). It was observed that Ypd1p has a significant stabilizing effect on phospho-Ssk1p; this effect is much less pronounced with a mutant version of Ypd1p that cannot be phosphorylated (256).…”

“…The entire reaction is, however, driven towards phosphorylation of Ssk1p, since the equilibrium of the reaction from Ypd1p His64 to Ssk1p Asp554 was on the side of the latter (256)(257)(258). It was observed that Ypd1p has a significant stabilizing effect on phospho-Ssk1p; this effect is much less pronounced with a mutant version of Ypd1p that cannot be phosphorylated (256). In vitro, the half-life of phospho-Ssk1p was about 13 min in the absence of Ypd1p, and this period increased 200-fold in the presence of Ypd1p.…”

“…These studies also demonstrate that in vivo the histidine kinase cannot bypass the Sln1p response regulator. Hence, Ypd1 does not normally become phosphorylated directly by the Sln1p histidine kinase (476), although this reaction can occur in vitro (256).…”

Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway

“…First, it has been reported that in the presence of a substoichiometric amount, Ypd1 extends the half-life of Ssk1ϳP dephosphorylation to ϳ42 h in vitro (13). However, how Ypd1 stabilizes a 10-fold excess of Ssk1ϳP or if a similar stabilization occurs in vivo is unknown.…”