Association of Yeast Adenylyl Cyclase with Cyclase-Associated Protein CAP Forms a Second Ras-Binding Site Which Mediates Its Ras-Dependent Activation

Abstract

Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiaeadenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras 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 Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras 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 Ras-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 Ras-binding site in effector regulation.

ACKNOWLEDGMENTS

We thank A. Seki and A. Kawabe for help in preparation of the manuscript.

This investigation was supported by grants-in-aid for scientific research on priority areas, for scientific research, and for JSPS fellows from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Diseases and from Sankyo Foundation of Life Science. F. Shima is supported by a fellowship from the Japan Society for the Promotion of Science.