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 crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1), Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Pas, we postulated that the associated CAP might contribute to the formation of a Pas-binding site of CYR1, which mediates CYR1 activation, other 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 defective in Pas signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified 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 the other CAP mutant, failed to be activated by Pas but exhibited a greatly enhanced binding to Pas, Conversely, a Pas mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Pas-binding site appears to be a critical determinant of CYR1 regulation by Ras: too weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Pas-binding site in effector regulation.