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    • br Introduction The Ras superfamily of small GTPases is comp

    2022-07-26


    Introduction The Ras superfamily of small GTPases is composed of more than 150 protein members involved in a wide variety of biological processes, such as proliferation, differentiation, cytoskeletal organization, and protein trafficking [1]. The Ras superfamily proteins act as molecular switches that cycle between GDP-bound inactive and GTP-bound active states to control the diverse biological processes. The rate of inactivation (transition from the GTP- to the GDP-bound state) is regulated by intrinsic GTPase activities of the small GTPases themselves and/or GTPase activities enhanced by GTPase-activating proteins (GAP). Conversely, the rate of activation (transition from the GDP- to the GTP-bound state) is determined by slow dissociation of GDP and subsequent fast loading of GTP to the small GTPases [2]. The picomolar affinity of GTP to the small GTPases and the 10-fold higher concentration of GTP over GDP in the cell environment make the GDP dissociation the rate-limiting step of the GDP/GTP exchange reaction. Guanine-nucleotide exchange factors (GEF) catalyze the GDP dissociation so that GTP can bind to a nucleotide-free GTPase. Thus, the intrinsic GDP/GTP exchange cycles of the small GTPases are regulated by two factors, the rate of GTP hydrolysis and the rate of GDP dissociation. Recently, a novel class of RAS superfamily GTPases, termed as ‘fast-cycling GTPases’, which do not follow the classical GDP/GTP exchange cycle has been proposed. This class of proteins, which currently includes 4 members of Rho family GTPases, RhoF, RhoD, RhoV, and RhoU, has a significantly elevated rate of GDP dissociation [3,4]. These fast-cycling GTPases have strong abilities to reactivate their GDP-bound states by rapidly releasing bound GDP. As a result, these GTPases can function as constitutively active GTP-bound proteins. One interesting molecular aspect of the fast-cycling GTPases is the fact that these proteins conserve most of the amino acids essential for GDP binding. For example, F28 and P29 residues (the amino closantel numbering of Rac1) have been shown to be essential for GDP binding, whose mutations accelerate the GDP dissociation in many RAS superfamily GTPases [[4], [5], [6], [7], [8]]. However, these residues are completely conserved in RhoF and RhoD, and replaced by similar amino acids in RhoV and RhoU (the phenylalanine residue is replaced by tyrosine). This fact indicates that the fast-cycling GTPases might accelerate the GDP dissociation through a yet unknown mechanism. Understanding the molecular basis of the fast-cycling GTPases is of biological significance, because it would provide novel insights into the mechanism of constitutive activation of RAS superfamily GTPases, which is involved in many pathological processes, including cancer and developmental syndromes [9,10]. These studies motivated us to investigate the GDP/GTP exchange cycle of RhoF GTPase. RhoF, or Rif, is a member of Rho GTPase family involved in a signaling pathway leading to filopodia formation [[11], [12], [13]]. A previous study using fluorescently labeled GDP suggested that RhoF is a member of the fast-cycling GTPases that dissociates GDP very rapidly (dissociation rate constant was 3.9 × 10−3 s−1) [4]. However, the study did not fully investigate the possibility of fluorescence artifacts and no other study has examined the GDP/GTP exchange cycle of RhoF. Moreover, the structural characteristic of RhoF, which may underlie the molecular basis of the fast-cycling, remains elusive. Here we generated a GTPase active recombinant RhoF and examined its structure and GDP/GTP exchange cycle.
    Materials and Methods
    Results
    Discussion The most striking result in this study was that the RhoF-GDP complex dissociates GDP very slowly with a dissociation rate constant of 1.3 × 10−6 s−1 (Fig. 3A). This value was approximately 103-fold slower when compared to the previously reported value for RhoF (3.9 × 10−3 s−1) [4]. The origin of such a large discrepancy remains unclear at this stage. Given that the GTPase activities of RhoF reported in the two studies are in a similar range (see Table 1 in Ref. [4]), this discrepancy likely arises from a difference in the fluorescence assay system. Either one of the studies might misassign a fluorescence artifact as the mGDP dissociation kinetics of RhoF. Accordingly, a further study using a different assay that does not rely on fluorescence is required to judge whether RhoF is an atypical fast-cycling or typical slow-cycling GTPase. However, we currently believe the latter, because the slow fluorescence decline, which we attributed to the true mGDP dissociation kinetics of RhoF, was accelerated by chelation of Mg2+ and by the canonical fast-cycling mutations, F44L and P45S (Fig. 3C). The markedly slow GDP dissociation kinetics of RhoF might be also a potential source of misassignment, because a typical mGDP dissociation experiment is conducted in a shorter time frame, up to ≈24 h [4], which hampers the detection of the extremely slow GDP dissociation.