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The evidence presented by Zhang et a l suggests a model for the relatively higher intrinsic rate constants for some Rho subfamily members over other members and other subfamilies. The involvement of the C-terminal tail which provides both a catalytic determinant, to cause an increase in hydrolysis, and a structural determinant, to allow protein-protein homodimerisation is a compelling one for the Rho subfamily members they investigate

(RhoC, Cdc42, Rac2, RhoA and RhoB), since it seems to concur with details known about the action of GTPase Activating Proteins. The conclusions they draw suggest an important activity for the C-terminal tail of Rho subfamily proteins, and this chapter describes an

investigation into the significance of the C-terminal tail of Racl.

The results described in section 3.4.1 and summarised in Table 1, show that^/WiWcj,; to the results presented by Zhang et a l, Racl shows little increase in rate constant as the concentration of either truncated Racl or full length Racl is increased. The increase observed in previous reports (up to 8-fold increase) is of a greater magnitude over measured concentrations, than observed in this report (up to 2.5-fold increase) These two sets of data [presented both in this report and by Zhang, B., et a l, show noj significant kinetic differences.

The presence of Rho subfamily GTP-analogues, such as Cdc42*GTPyS was shown to increase the GTP hydrolysis rate constant and to allow the formation of homodimers (Zhang, B., et a l 1999). The C-terminal tail was suggested to be of importance in these activities. In this report GTP-analogues (Racl • GMPPNP) of both the truncated Racl and fiill length Racl protein were used to investigate this property of Rho subfamily proteins in their active states. Incubation of truncated forms of Racl* GMPPNP and Racl * GTP, would not be expected to show an increase in rate constant, since they lack the C-terminal tail necessary for any increase in GTP hydrolysis. On the other hand, full length forms of Racl • GMPPNP and Racl • GTP, which contain the C-terminal tail, would be expected to show some form of increased GTP hydrolysis as the concentrations of Rac* GMPPNP were increased. The results presented in section 3.4.2 show that, both truncated and full length Rac*GTP do not show any increase in rate constant on the addition of truncated or Rac * GMPPNP full length

The incubation of the C-terminal peptides, as described in section 3.4.2, with full length

Rac*GTP would also be expected to provide the C-terminal determinants (as suggested by Zhang, B., et a l ) and show an increase in GTP hydrolysis — only if the C-terminal tails are necessary and sufficient for the stimulatory activity. The presence of these peptides made a negligible difference on the rate constants observed for full length Racl.

The investigations described in this report, using Racl, show that the measured GTPase

rates do not differ significantly fi'om those measured by Zhang, B., et a l

The rate constants observed in this study for Racl and those measured in Zhang, B., et a l,

are shown in Table 1. This table of data shows that for related proteins, such as Racl and

Rac2, the difference in rate constants is not significant. Although small difierences between

the data exist, the overall data is within an order of magnitude and therefore consistent

with each other. The differences that are present in the data could be due to temperature

effects — this study measures at 30 °C, Zhang, B., et a l at 23 °C — or due to the different

methodology used to measure the GTP hydrolysis. Previous measurements of Rad’s

intrinsic GTP hydrolysis at different temperatures, show rate constant for GTP hydrolysis of

4.8 X 10'^ s'^ (20 °C) and 6.5 x 10^ s'^ (37 °Q (Bernard, A., et a l 1992), which is consistent

with the data presented here. The MESG system used by Zhang, B., et a l has been used

previously (Webb, M., and Hunter, J., 1992) and provided data that is also consistent with

the data presented here and in Zhang, B., et a l The slight differences between the data

presented here and in Zhang, B., et a l may be due to variations in the actual protein

concentrations present, or variations in the active protein concentration, as well as

variations in the efficiency of the GTP nucleotide loading onto Rac. From the data

presented here Racl does indeed have a higher GTPase hydrolysis compared to other Rho

subfamily proteins, although the importance of the C-terminal tail in this process is

unclear.

Zhang, B., et a l have suggested that it is a specific Arg residue involved in this stimulation of catalysis, in a similar fashion to the process used by GAPs to promote catalysis. It has been shown that this ‘Arg finger’ is not the only residue involved in stimulation of GTP hydrolysis by GAPs (Sermon, B., et a l 1988) and so the ability of a single Arg residue, present in the C- terminal tail, to stimulate GTP hydrolysis to levels similar achieved by a complete GAP molecule would be quite unlikely.

The presence of bound nonhydrolysable GTP analogues, such as GTP-yS, were shown to increase the rate of hydrolysis of RhoC, Rac2 and Cdc42 to that almost equivalent to the rate of G-proteins in the presence of an active GAP (8-folc^, and this current study

demonstrated GTP hydrolysis levels which were consistent with previous work. ' ■ The hypothesis that Rho subfamily-bound GTP analogues are activatory via their C-terminal tail, suggests that the C-terminal tail becomes activated in some way in the active state. It is possible that in a GDP-bound form the C-terminal tail is sequestered by the G-protein

preventing its activatory ability. In the GTP-bound form the C-terminal tail somehow becomes active, and is able to perform its putative role. Both crystal and NMR structures have shown that in both GDP- and GTP- forms of Rho subfamily proteins, the C-terminal tail is essentially very mobile and unstructured. This suggests that there can be little or no change in C-terminal tail structure or molecular-associations between the two states of the proteins. This further suggests that both GDP-bound and GTP-analogue-bound forms of Rho subfamily proteins would be able to stimulate GTP hydrolysis of susceptible Rho subfamily proteins (such as Rac2). The GDP forms of the proteins were not observed to have this

ability by Zhang, B., et a l

The presence of the C-terminal peptides had no effect on the rate of GTP hydrolysis,

which may be expected if the C-terminal tails are not directly involved in the dimérisation

process (but are involved in hydrolysis acceleration), or if certain structural characteristics

are required in the C-terminal tail, which would almost certainly not be present in isolated

peptides in solution.

It is possible that both active and inactive forms of certain Rho subfamily proteins are able

to homodimerise through a separate region of the protein, \\diich brings the C-terminal tail

into proximity of the catalytic site. Indeed, the Cdc42*GDP form is able to homodimerise

as shown by column immobilisation studies, although shows no activatory activity

suggesting that the dimérisation and activatory characteristics may be separate regions of

the protein.

Observations in this laboratory (Webb, M., personal communication) have suggested that truncated Racl • GDP is able to exist in both monomeric and homodimeric forms, possibly in some form of conformational equilibrium. The observation of Racl*GDP being able to

dimerise, without a C-terminal tail, further suggests that the C-terminal tail may not play a

significant role in Rac dimérisation. However, the C-terminal tail’s role in accelerating the

GTP hydrolysis of Racl cannot be discounted fix>m the work presented in this present

study.

Chapter 4