Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11.
BACKGROUND
In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex. Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling.


RESULTS
A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11. The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases. Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains.


CONCLUSIONS
Pheromone induces binding of Gbeta to Ste5 through its LIM domain. This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of Gbeta.
Feng
Y
Y
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Song
L Y
LY
Kincaid
E
E
Mahanty
S K
SK
Elion
E A
EA
D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002352:Carrier Proteins; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010641:Phenotype; D017354:Point Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D015398:Signal Transduction; D014157:Transcription Factors
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D005656:Fungal Proteins; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C071596:ste11 protein, S pombe; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C075580:FUS3 protein, S cerevisiae; C541984:KSS1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D000409:Alanine