The DH and PH Domains of Trio Coordinately Engage Rho GTPases for their Efficient Activation

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Abstract

Rho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid-binding regions, they also participate in protein–protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is fourfold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in engaging Rho GTPases directly for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains.

Introduction

Rho GTPases act as molecular switches that cycle between inactive GDP-bound states and active GTP-bound states.1., 2., 3. Once activated, Rho GTPases bind to their effectors to elicit a variety of downstream signaling responses, including: cytoskeletal reorganization, gene expression, cell cycle progression, membrane trafficking, cell adhesion, and cell migration.3., 4. Given their involvement in many critical cellular processes, it is not surprising that aberrant regulation of Rho GTPases contributes to various diseases such as cancer,5 hypertension,6 and mental retardation.7 It is generally appreciated that delineating the mechanisms involved in the activation of Rho GTPases might guide treatment regimens for a variety of human diseases.

The activity of Rho GTPases is regulated mainly by three classes of proteins: (1) GTPase activating proteins (GAPs) accelerate the hydrolysis of bound GTP; (2) guanine nucleotide dissociation inhibitors (GDIs) sequester prenyl groups added post-translationally to Rho GTPases and thus stabilize cytosolic, inactive forms of the GTPases; and (3) Dbl-family guanine nucleotide exchange factors (GEFs) catalyze the exchange of bound GDP for GTP, thereby activating the GTPases.1., 8., 9., 10.

Not including splice variants, the human genome encodes 69 Dbl-family GEFs10 of varying domain architecture, size, and GTPase specificity.1., 10. All Dbl-family members contain a conserved Dbl homology (DH) domain, followed almost invariably by a tandem pleckstrin homology (PH) domain.2., 10. The DH domain forms the majority of the interface with the GTPase and is often sufficient to catalyze nucleotide exchange,10., 11., 12. and to dictate GTPase specificity.10., 11., 13. The PH domain is necessary for regulating exchange in vivo,14., 15., 16., 17., 18. and in many cases, in vitro,13., 19., 20., 21., 22. but its exact functions are unclear.

DH-associated PH domains, like most PH domains, are traditionally characterized as phosphoinositide-binding modules; but the ability of phosphoinositides to allosterically regulate the exchange activities of Dbl-family GEFs is, at best, controversial.16., 18., 23., 24., 25. In addition, in several instances, DH-associated PH domains are not necessary for recruiting GEFs to cellular membranes where they normally operate on membrane-resident GTPases. This is true even though PH domain mutations abrogating phosphoinositide binding diminish the ability of Dbl-family GEFs to activate their cognate GTPases in vivo.16., 18., 26., 27. Another possibility that is finding growing support is that DH-associated PH domains facilitate exchange through direct interactions with cognate GTPases.13., 19., 20., 26., 28. For example, structures of Dbs DH/PH in complex with nucleotide-depleted Cdc42 or RhoA (henceforth, Dbsradical dotCdc42 or Dbsradical dotRhoA, respectively) show direct contacts between the β3/β4 loop of the PH domain and its cognate GTPases.13., 28. Mutational analysis has confirmed the catalytic importance of this interface both in vitro and in vivo, and has identified His814, Gln834, and Tyr889 of Dbs as functionally significant for guanine nucleotide exchange.18., 28. Given the highly conserved position of PH domains directly adjacent to DH domains, it is likely that other Dbl-family GEFs also use their DH-associated PH domains to engage their cognate GTPases directly to facilitate guanine nucleotide exchange. In particular, published data on the Dbl-family GEF Trio22., 29. and the Caenorhabditis elegans ortholog of Trio, UNC-73,26 provide strong circumstantial support for this hypothesis.

Trio is a large, evolutionarily conserved Dbl-family GEF that is best characterized for its role in regulating neurite outgrowth.30., 31., 32., 33., 34., 35., 36., 37. Unlike most Dbl-family GEFs, Trio has two DH/PH cassettes (Figure 1(a)); the first DH/PH cassette catalyzes exchange on Rac1 and RhoG,38., 39., 40., 41. while the second DH/PH cassette is specific for RhoA.38., 39. Physiological functions of the C-terminal DH/PH cassette remain relatively unclear, but the N-terminal DH/PH cassette is critical and often sufficient for regulating neuronal development through activation of RhoG, and possibly Rac1.31., 35., 36. Interestingly, although there is some discrepancy over the relative rates of exchange, the isolated N-terminal DH domain of Trio does not exchange as effectively as the corresponding DH/PH cassette in vitro.22., 29. Furthermore, deletion of the PH domain from the N-terminal DH/PH cassette of full-length Trio reduces its ability to induce neurite outgrowth in PC-12 cells through activation of RhoG.29 Finally, residues in the PH domain of Dbs that are functionally significant for exchange on its cognate GTPases are conserved in Trio (Figure 1(b)). Together, these data suggest strongly that the N-terminal DH/PH cassette of Trio might use its PH domain similarly to Dbs when catalyzing exchange on its cognate GTPases Rac1 and RhoG.

Thus, to better understand the possible functional interplay between the N-terminal DH and PH domains of Trio specifically, and between DH and PH domains in general, we determined the crystal structure of the N-terminal DH/PH cassette of Trio in complex with nucleotide-depleted Rac1 (henceforth, Trioradical dotRac1). The complex recapitulates many of the interactions involving the PH domain previously seen in the Dbs/GTPase structures that are required for efficient guanine nucleotide exchange by Dbs. Mutation of this interface in Trio confirms the necessary involvement of the N-terminal PH domain of Trio in engaging Rac1 and RhoG directly for their efficient activation in vitro and in vivo. These studies further support the general capacity of DH-associated PH domains to be active participants in the exchange process and suggest a coherent model of guanine nucleotide exchange catalyzed by Dbl-family proteins that requires the direct and cooperative engagement of DH domains, as well as their associated PH domains, by Rho GTPases for their efficient activation.

Section snippets

Structure of Trio in complex with nucleotide-depleted Rac1

A complex between the N-terminal DH/PH cassette of Trio (residues 1226–1536) and residues 1–189 (C189S) of Rac1 yielded poorly diffracting crystals. Since several structures of Rho GTPases indicate that the C-terminal polybasic tail is not typically well structured, nor involved directly in binding GEFs,13., 19., 28., 42. a truncated version of Rac1 lacking this region and ending at residue 177 (Rac177) was crystallized with the N-terminal DH/PH cassette of Trio and used for structure

Discussion

The work presented here describes specific interactions between the N-terminal PH domain of Trio and Rac1 or RhoG that are essential for productive activation of these GTPases. These interactions strikingly mimic the coordinate engagement of RhoA or Cdc42 by the DH and PH domains of Dbs.13., 28. Moreover, PH domain-mediated interactions between both Trio and Dbs, and their cognate GTPases, are required for efficient guanine nucleotide exchange in vitro and in vivo and indicate that the

Sequence alignment

The sequences of Dbs (NP_079255), Trio (NP_009049), Duo (NP_003938), Dbl (NP_005360), Duet (NP_008995), PRex1 (NP_065871), p63RhoGEF (NP_891992), Lfc (NP_004714), neuroblastoma (NP_005263), and obscurin (NP_443075) shown in Figure 1(b) were aligned using CLUSTAL_X.55 The NCBI accession numbers are given in parentheses.

Protein preparation for guanine nucleotide exchange assays

Human Trio DH/PH (residues 1226–1535) (kindly provided by Dr Yi Zheng, Cincinnati Children's Hospital Medical Center ) was encoded as a fusion with an N-terminal His6 tag in

Acknowledgements

We acknowledge Dr Ashutosh Tripathy at the UNC-Chapel Hill Macromolecular Interactions Facility for his assistance with circular dichroism spectroscopy. We acknowledge Dr Alex Singer for his technical assistance with model building and refinement. This work was supported by National Institutes of Health grants GM62299 and GM65533.

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