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

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

F Shima et al. Mol Cell Biol. 2000 Jan.

Abstract

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.

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Figures

FIG. 1

Posttranslational modification of Ras is required for association with the CYR1-CAP complex but not for association with the LRR domain. (A) The modified and unmodified forms of Ha-Ras were loaded with GTPγS (GTP) or GDPβS (GDP) and incubated at 150 nM with approximately 0.1 μg of GST-CYR1(606–1764), which had been purified from yeast TS5 cells (Table 1) and immobilized on glutathione-Sepharose resin as described in Materials and Methods. The bound proteins were eluted with buffer A containing 20 mM glutathione, and GST-CYR1(606–1764) and Ha-Ras in the eluate were detected by Western immunoblotting with anti-GST polyclonal antibody (lower panel) and anti-Ha-Ras monoclonal antibody F235 (upper panel), respectively. (B) A complex of GST-CYR1(1764–2026) and FLAG-CAP, purified from yeast FS66 cells, and GST-CYR1(1764–2026), purified from E. coli, were immobilized on glutathione-Sepharose resin. FLAG-CAP, purified from yeast FS20 cells, was immobilized on anti-FLAG M2 resin. Aliquots (40 μl) of the resin carrying the various proteins were subjected to the in vitro binding assays with Ha-Ras as described in panel A. The top panels show the bound Ha-Ras. The middle panels show FLAG-CAP (approximately 0.1 μg) copurified with GST-CYR1(1764–2026), which was detected with anti-FLAG antibody (Kodak). The bottom panels show GST-CYR1(1764–2026) (approximately 0.4 μg). The experiments were repeated five times, yielding similar results.

FIG. 2

Screening for CAP mutants defective in Ras signaling but retaining the ability to associate with CYR1.

FIG. 3

In vivo function of CAP mutants obtained by the screening. (A) Heat shock sensitivity of yeast cells expressing the various CAP mutants. TK161-R2V(CAPΔN) yeast cells harboring pGBT10-CAP(1–77) carrying the indicated mutations were examined for heat shock sensitivity by a replica-plating method as described in Materials and Methods. Shown are photographs of the two replica plates, one subjected to a 55°C heat shock for 5 min (left panel) and the other without the heat shock treatment (right panel), after 2 days of growth at 30°C. (B) Yeast two-hybrid analysis for interaction of CAP(1–77) carrying the indicated mutations with CYR1(1898–2026) was performed as described in Materials and Methods. (C) FLAG-CAP carrying the indicated mutation was purified from yeast strains FS25, FS55, FS56, or FS57 (Table 1) as described in Materials and Methods. The endogenous CYR1, which was copurified with the FLAG-CAP mutants, was subjected to Western immunodetection. The upper panel shows the endogenous CYR1 detected with anti-CYR1CT antibody. The lower panel shows purified FLAG-CAP proteins (approximately 0.5 μg) detected with anti-FLAG antibody. All of the experiments were repeated three times and yielded similar results.

FIG. 4

In vitro activation of CYR1 complexed with the CAP mutants by the modified Ras. The endogenous CYR1 complexed with the FLAG-CAP mutants was purified as described in the legend to Fig. 3C and examined in vitro for stimulation of adenylyl cyclase activities by the GTPγS-bound forms of the modified (A) or the unmodified (B) Ha-Ras as described in Materials and Methods. The y axis shows the Ras-dependent adenylyl cyclase activity, which is presented as a ratio to the Mn2+-dependent activity of the same specimen. The Mn2+-dependent activities of the purified proteins ranged from 15 to 20 pmol of cAMP formed during 30 min of incubation at 30°C. Similar experiments performed on three occasions with different preparations of CYR1-CAP complex yielded equivalent results.

FIG. 5

In vitro association of the CYR1(1764–2026)-mutant CAP complex with Ha-Ras. GST-CYR1(1764–2026) complexed with FLAG-CAP carrying the indicated mutations was purified from yeast strains FS66, FS68, FS69, and FS70 and examined for in vitro association with the modified and the unmodified forms of Ha-Ras as described in the legend to Fig. 1B. The top panel shows the bound Ha-Ras, and the middle panel shows FLAG-CAP (approximately 0.05 μg) copurified with GST-CYR1(1764–2026). The bottom panel shows GST-CYR1(1764–2026) (approximately 0.2 to 0.4 μg) eluted from the resin. The experiments were repeated three times, yielding equivalent results.

FIG. 6

H-Ras(E31K) is incapable of activating CYR1 efficiently and exhibits a greatly enhanced binding to the CYR1-CAP complex. (A) The full-length CYR1 expressed in yeast TK36-1 cells (Table 1) was solubilized from the membrane fraction and was examined for activation by the modified and the unmodified forms of wild-type Ha-Ras (WT) and by the modified form of Ha-Ras(E31K) (E31K), all in the GTPγS-bound configurations, as described previously (43). One unit of activity is defined as 1 pmol of cAMP formed in 1 min of incubation with 1 mg of protein at 30°C. (B) The association of the indicated forms of Ha-Ras with GST-CYR1(606–1764) was examined in vitro as described in the legend to Fig. 1A. (C) The association of the indicated forms of Ha-Ras with GST-CYR1(1764–2026) complexed with FLAG-CAP were examined in vitro as described in the legend to Fig. 1B. Similar experiments performed on two occasions with different preparations of CYR1 and Ha-Ras yielded equivalent results.

FIG. 6

FIG. 7

Model of interaction among CAP, CYR1, and Ras. Spinning-wheel representations of the mutually interacting segments of CAP and CYR1. The amino acids corresponding to the a, b, and g positions of CAP(1–36) and to the a and d positions of CYR1(1916–1940) are shown. The residues of CAP and CYR1 whose mutations abolished the CAP-CYR1 interaction are shown in italic type (data were taken from reference 37). The residues of CAP whose mutations resulted in gross alteration of the association with the modified Ha-Ras are shown in boldface type. The broken lines indicate possible interactions predicted from the mutational studies. See Discussion for a detailed explanation.

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