Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

Abstract

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 sub-branch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domains. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively-active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1 Δ cells, whereas robust Hog1 activation occurred in AHK1 + cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 sub-branch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross-talk. Thus Ahk1 is a scaffold protein in the HKR1 sub-branch, and prevents incorrect signal flow from Hkr1 to Kss1.