Discusión de resultados

To gain further support that the mutants could bind to R acl, direct physical interaction between R acl and GST fusion proteins of the wild-type and n-chimaerin carboxyl- terminal region mutant proteins, which had been immobilized onto glutathione Sepharose columns, was examined. When purified thrombin-cleaved Q61LRacl.[y- ^^P]GTP was loaded onto a glutathione Sepharose column containing GST/wild-type carboxyl-terminal region n-chimaerin protein, the R acl protein was eluted by about 1.5x the dead volume of the column (Fig. 33). In contrast, Q61LRacl.[y-^^P]GTP co­ eluted with the dead volume of a glutathione Sepharose column immobilized with control GST protein (Fig. 33). The differential elution patterns of Q61LRacl.[y- ^^P]GTP on the two columns confirmed that Q61LRacl.[y-^^P]GTP binds to the carboxyl-terminal region o f n-chimaerin. When Q61LRacl.[y-^^P] was next loaded onto a glutathione-Sepharose column immobilized with GST fusion protein o f mutant 107, the R acl protein was also retained on the column and co-eluted with about 1.8x the dead volume o f the column (Fig. 33). This result provided direct support for the ability of mutant 107 to bind R acl.

Fig. 32 Competitive inhibition of n-chimaerin Racl GAP activity by mutants 107, 108 and 109

The R acl GAP competition assay was performed as described in section 6.4 o f the Materials and Methods chapter. The competitors were mutants 107 (o ), 108 (■) and 109 (°). For comparison, data points corresponding to competitive inhibition o f n- chimaerin carboxyl-terminal region protein using wild-type Racl.GTPyS ( • ) has been included. The result is representative of two separate experiments.

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Fig. 33 Interaction of Q61LRacl with wild-type and mutant if-chimaerin carboxyi- temiinal region proteins on columns

Interaction o f Q 6 1 LRac 1.[y-^^P]GTP with wild-type n-chimaerin carboxyl-terminal region protein (GST/C) (o ), mutant 107 ( • ) and GST (a) were examining by monitoring the differential retention of the labelled p21 on glutathione columns immobilized with the mentioned proteins. The elution volume for the p21 has been expressed as a fraction of the dead volume of each column used. Hence a value o f 1 indicates that the labelled p21 was eluted by a volume equivalent to the dead volume of the column. The amount of [y-^^P]GTP remaining bound to R acl in each fraction is expressed as counts per minute (c.p.m.). Binding of [y-^^P]GTP to R acl was determined by rapid filtration as described in the GTPase assay (see section 6.1 o f the Materials and Methods chapter). Similar results were obtained in another experiment.

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5. Biochemical characterization o f R a cl mutants

M utation of Gln61 to Leu in Ras increases the affinity for p i 20-GAP by approximately 10-fold as compared with wild-type or G12VRas (Bollag and M cCormick, 1991). As shown previously in Fig. 31, an approximately 10-fold higher affinity for GAP (i.e. n-chimaerin) was also observed with R acl (Q b lL R acl) containing the equivalent mutation as compared with wild-type or G 12V Racl. The GTPase activity o f the Q blLR as mutant is thought to be insensitive to GAP because Gln61 has been postulated to be involved in the catalytic reaction (Gideon et a l ,

1992). To ascertain if this characteristic was similar for the equivalent R acl mutant, the GTPase activity o f Q blL R acl was measured in the presence and absence o f n- chimaerin. Compared to an intrinsic GTPase activity of wild-type R acl which resulted in approximately 75-80% GTP remaining bound to R acl after 10 min at 15°C, the GTPase activity o f Q blL R acl was significantly lower (95-100% GTP remaining bound) when measured over the same period (Fig. 34). Binding to [y-^^P]GTP by Q b lL R acl was, however, similar to wild-type and hence unaffected by the mutation. Addition of «-chimaerin to Q61 LRacl.[y-^^P]GTP did not affect the mutant, suggesting that Q b lL R acl has similar characteristics as Q blLR as. Fig. 34 also shows that constitutively activated G12VRacl has a reduced GTPase activity compared with wild-type R acl which was insensitive to «-chimaerin.

Substitution of Asn for Thr at position 17 of R acl yields a dominant negative inhibitory protein (T17NRacl) whose expression in cells interferes with endogenous R acl function (Ridley et al., 1992). Inhibtion by T17N Racl is thought to be due to its action as a competitive inhibitor of wild-type R acl for interactions with a nucleotide releasing factor. To determine whether «-chimaerin was active on the T17N R acl mutant, the GTPase activity of T17NRacl was initially measured in the absence of GST/«-chimaerin carboxyl-terminal region protein. The T17N Racl mutant was observed to bind approximately 3-6% [y-^^P]GTP as compared with wild-type or G 12V Racl. This finding suggested that T17N Racl, like S17NRas, may display a preferential affinity for GDP than GTP (Feig and Cooper, 1988). To examine this possibility, the binding affinities of T17N- and wild type-Racl proteins for GDP and

GTP were determined. Although both proteins bound GDP with the same affinity (IQ = 20-40 nM), the affinity for GTP differed (Fig. 35). T17N- and wild-type R acl bound GTP with IQ values of 0.3 pM and 0.02 pM respectively. This approximately 10-fold preferential binding affinity of TlTN R acl for GDP rather than GTP is consistent with the findings o f Feig and Cooper (1988) as well as Ridley et al. (1992). Throughout the 10 min of the assay, no significant change in the amount o f [y- ^^P]GTP bound to TlTN R acl was observed (Fig. 34). Addition of 0.1 pM GST/«- chimaerin carboxyl-terminal region protein, which stimulated the GTPase activity of wild-type R acl as represented by a decrease of tgo% from 12 to 5 min, did not affect the GTPase activity o f TlT N R acl (Fig. 34).

Fig. 34 GTPase activities of wild-fype, G12V-, T17N- and Q61LRacl in the presence and absence of /i-chimaerin

Intrinsic GTPase activity o f (a) G12V- (o ), (b) Q61L- (a), (c) T17N- (0) and (d) wild- type (°) R acl was measured as described in section 6.1 of the Materials and Methods chapter. GTPase activity of G12V- ( • ) , Q blL R acl (^), T17N- (♦) and wild-type (■) R acl was also measured in the presence of 0.1 pM GST/n-chimaerin carboxyl-terminal region protein (GST/C). The result shown below is representative of two experiments. The initial binding o f [y-^^P]GTP by G12V-, Q61L- and wild-type R acl was o f a similar level and approximately 200000-30000 c.p.m.. However, the initial binding of [y_32p]GTP by T17N Racl was only about 3-6% that o f G12V-, Q61L- and wild-type R acl.

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