TABLE 1. Activity of p63RhoGEF DH/PH variants and their affinity for Gαi/q.
Fold GEF activation is the rate of 400 nM p63RhoGEF DH/PH domain catalyzed guanine nucleotide exchange on RhoA divided by the intrinsic exchange rate of RhoA, as measured by rate of change in FP of BODIPY FL GTPγS as it binds RhoA. The average apparent rate constant for these experiments was 0.014 ± 0.002 min−1 (n=10). Fold Gαi/q activation is the exchange rate of 400 nM DH/PH domains in the presence of 800 nM Gαi/q divided by that of the DH/PH domain alone. The inhibition constant for each variant (KI) was measured using FCPIA to monitor competition with 100 nM AF-labeled DH/PH binding to bead-bound biotinylated Gαi/q.
| P63RhoGEF variant |
Fold GEF activation |
Fold Gαi/q activation |
KI (nM) |
|---|---|---|---|
| DH/PH-wt | 1.4 ± 0.13 (10) | 3.5 ± 0.70 (10) | 46 ± 14 (9) |
| DH/PH-P330L | 1.1 ± 0.07 (4) | 1.2 ± 0.03 (4) | 40. ± 6.9 (3) |
| DH/PH-M336S | 1.6 ± 0.17 (4) | 2.2 ± 0.16 (4) | 68 ± 29 (3) |
| DH/PH-G340I | 2.6 ± 0.43 (3) | 17 ± 9.1 (3) | 670 ± 490 (4) |
| DH/PH-R341A | 2.2 ± 0.25 (5) | 7.2 ± 1.9 (5) | 75 ± 33 (4) |
| DH/PH-R341E | - | - | 120 ± 38 (3) |
| DH/PH- Δ(397-402) | 1.3 ± 0.13 (4) | 4.0 ± 0.54 (9) | 48 ± 8.3 (3) |