Signaling in the Yeast Pheromone Response Pathway: Specific and High-Affinity Interaction of the Mitogen-Activated Protein (MAP) Kinases Kss1 and Fus3 with the Upstream MAP Kinase Kinase Ste7

Abstract

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.

Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates

“…This method may be prone to some bias for fusion proteins that do not transfer to the immunoblot membrane as efficiently as the standard; this would lead to an underestimation of the cellular concentrations of poorly transferring proteins. Nevertheless, for Ste7 MEK , Fus3 MAPK and Kss1 MAPK , this method gave estimates in good agreement with those obtained using native test proteins and standards produced by in vitro translation [25].…”

“…K d estimates should be calculated by applying the above formula to the results of multiple replicate experiments and averaging the results [25,30]. An example is shown in Table 2.…”

“…D-sites (also called 'D-domains', 'DEJL motifs', or 'kinase interaction motifs') were first identified in the yeast MEK Ste7 and the transcription factors c-Jun and Elk-1 [25,[47][48][49], and were subsequently found in numerous MAPK-interacting proteins. A second docking motif (consensus L-X 1-2 -(R/K) [2][3][4][5] ), related to the D-site, is found in MAPK-activated kinases such as RSK1 [50].…”

“…This is facilitated by making the fragments by in vitro translation. An example of such a procedure can be found in Bardwell et al [25] and Molina et al [32]. In the former example, the use of progressively smaller N-terminal fragments of Ste7 MEK eventually led the authors to identify a highaffinity D-site at the very N-terminus of Ste7 [25].…”

“…In this paper, we summarize methods we have used successfully and repeatedly to detect MAPK activation in cells [21][22][23][24], estimate the cellular concentrations of MAPKs and other proteins of interest [25]; detect and quantify the stable physical association of MAPKs with their substrates and regulators [24][25][26][27][28][29][30][31][32], and delineate the MAPK-binding regions or domains of MAPK-interacting proteins [24][25][26][29][30][31][32].…”