Suppose you are studying a kinase signaling cascade using a yeast model system. It has been determined that the key output signal of the cascade is production of a rare phosphorylated plasma membrane lipid, phosatidylinositol 3-phosphate (PI-3-P), which is simply PI that has been phosphorylated at the 3-position on the inositol head-group. You have identified three genes that specifically affect production of this signal, called pik1, pik2, and pik3. All three genes are predicted to be kinases. The problem is that kinases in this family can either be protein kinases or lipid kinases, this cannot be determined from the sequence, and you need to know which one is the lipid kinase that produces PI-3-P.

(1) Your screen produced knockout alleles of all three kinases. The phenotype of the pik1 mutant and pik3 single mutants was decreased production of PI-3-P. The phenotype of the pik2 mutant was over-production of PI-3. You also made and tested the following double mutant strains: pik1+pik2, increased PI-3-P production; pik2+pik3, decreased PI-3-P production. Which gene encoded the PI-3 kinase and how do you know?

(2) Speculate on the roles of the other two kinases, what does each of their phosphorylations do to the activity of their targets?