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Molecular and Cellular Biology, January 2000, p. 26-33, Vol. 20, No. 1
Department of Physiology II, Kobe University
School of Medicine, Chuo-ku, Kobe 650-0017, Japan
Received 11 August 1999/Returned for modification 9 September
1999/Accepted 1 October 1999
Posttranslational modification, in particular farnesylation, of Ras
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 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.
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Association of Yeast Adenylyl Cyclase with
Cyclase-Associated Protein CAP Forms a Second Ras-Binding Site Which
Mediates Its Ras-Dependent Activation
*
Corresponding author. Mailing address: Department of
Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-382-5380. Fax:
81-78-382-5399. E-mail: kataoka{at}kobe-u.ac.jp.
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