Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation

Posttranslational modification, in particular farnesylation, of Pas is cruc ial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1), Bas ed on the previous observation that association of CYR1 with cyclase-associ ated protein (CAP) is essential for its activation by posttranslationally m odified Pas, we postulated that the associated CAP might contribute to the formation of a Pas-binding site of CYR1, which mediates CYR1 activation, ot her than the primary Pas-binding site, the leucine-rich repeat domain. Here , we observed a posttranslational modification-dependent association of Pas with a complex between CAP and CYR1 C-terminal region. When CAP mutants de fective in Pas signaling but retaining the CYR1-binding activity were isola ted by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by mo dified Pas and exhibited a severely impaired ability to bind Pas, providing a genetic evidence for the importance of the physical association with Pas at the second Pas-binding site. On the other hand, CYR1, complexed with th e other CAP mutant, failed to be activated by Pas but exhibited a greatly e nhanced binding to Pas, Conversely, a Pas mutant E31K, which exhibits a gre atly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 effi ciently. Thus, the strength of interaction at the second Pas-binding site a ppears to be a critical determinant of CYR1 regulation by Ras: too weak and too-strong interactions are both detrimental to CYR1 activation. These res ults, taken together with those obtained with mammalian Raf, suggest the im portance of the second Pas-binding site in effector regulation.