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

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 response regulator, several two-component systems utilize additional means of dephosphorylation. For example, in some cases the sensor histidine kinase exhibits phosphatase activity towards its cognate response regulator (12, 13, 21, 30, 31, 35). Alternatively, an auxiliary protein may act to accelerate or catalyze dephosphorylation of the response regulator (10, 24, 33). Another mechanism for signal decay has been identified in the Escherichia coli Arc system, whereby dephosphorylation of the cytoplasmic response regulator occurs through a “reverse” phosphorelay which is dependent on the C-terminal HPt domain of ArcB (9). The osmoregulatory pathway in Saccharomyces cerevisiae consists of a multistep phosphorelay system involving the transmembrane hybrid histidine kinase SLN1, the HPt protein YPD1, and the response regulator SSK1 (36). In contrast to most two-component systems, these proteins are maintained in a phosphorylated state under normal environmental conditions. Under hyperosmotic stress conditions, SSK1 is rapidly dephosphorylated by a mechanism not well understood and in its dephosphorylated form activates a downstream mitogen-activated protein kinase cascade (28). The only other known response regulator in S. cerevisiae, SKN7, is also at least partially dependent on phosphorylation via SLN1 and YPD1 (18, 20). The multifunctional SKN7 protein has been implicated in maintenance of cell wall integrity (3), G1 cyclin expression (2, 23), and responses to oxidative and osmotic stress through a phosphorylation-dependent (SLN1-YPD1) as well as a phosphorylation-independent pathway (18–20, 22). Thus, YPD1 is required for phosphoryl group transfer between all three known response regulator domains in S. cerevisiae (i.e., SLN1-R1, SSK1-R2, and SKN7-R3). We previously reported the half-lives of the phosphorylated response regulator domains associated with the yeast osmoregulatory proteins SLN1 and SSK1, which were determined, due to experimental constraints, in the presence of substoichiometric amounts of YPD1 (15). We have since developed a means for phosphorylating these response regulator domains that does not rely on the presence of YPD1. We were thus able to determine what effect the presence of YPD1 has on the phosphostability of the response regulator domains. Our results indicate that the HPt protein YPD1 has a dramatic stabilizing effect on phospho-SSK1-R2 but not phospho-SLN1-R1 or phospho-SKN7-R3. We believe that this effect is mediated, at least in part, by the formation of a stable HPt-response regulator domain complex.