A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

MAP kinase dynamics in response to pheromones in budding yeast.

Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood. During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus.

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.