Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo.
BACKGROUND
Many signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades. Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs. It is not understood how MEKKs are regulated by extracellular signals. In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation. Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro.


RESULTS
Ste20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms. Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner. The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones. Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain.


CONCLUSIONS
Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.
Drogen
F
F
Swiss Institute for Experimental Cancer Research (ISREC), Switzerland.
O'Rourke
S M
SM
Stucke
V M
VM
Jaquenoud
M
M
Neiman
A M
AM
Peter
M
M
D002453:Cell Cycle; D003433:Crosses, Genetic; D005656:Fungal Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction
C490024:FUS1 protein, S cerevisiae; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; C067018:Ste11 protein, S cerevisiae