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Previous Article | Next Article 
Molecular and Cellular Biology, October 2003, p. 6823-6835, Vol. 23, No. 19
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.19.6823-6835.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Samuel Lunenfeld Research Institute of Mt. Sinai Hospital, Toronto, Ontario M5G 1X5,1 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada2
Received 16 May 2003/ Returned for modification 17 May 2003/ Accepted 1 July 2003
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ABSTRACT |
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INTRODUCTION |
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Guanine nucleotide exchange factors (GEFs) control the levels of functionally active GTP-bound, versus inactive GDP-bound, GTPases by stimulating the exchange of GDP for GTP (25, 41). Most proteins with demonstrated in vitro GEF activity for Rho, Rac, or Cdc42 contain a conserved Dbl homology (DH) domain of approximately 200 residues, which, in a number of cases, is sufficient to catalyze the exchange of GDP for GTP in vitro (23).
Regulation of GEF activity can occur through a number of different mechanisms. For example, the Vav family of Rho/Rac exchange factors can be phosphorylated on tyrosine 174 by Lck (12), resulting in activation of GEF activity through a mechanism involving displacement of the phosphorylated peptide motif from the DH domain (1). Tiam1 demonstrates specific activity toward Rac, which is enhanced by kinases, such as protein kinase C and Ca2+/calmodulin-dependent protein kinase II (19).
The DH domains of such proteins are typically followed by an adjacent pleckstrin homology (PH) domain of 
100 residues, a signaling module that is often involved in intracellular membrane targeting. The characteristic pairing of DH and PH domains suggests that the DH-associated family of PH domains may participate in the regulation of GEF activity. PH domains could contribute binding or catalytic residues, allosterically influence catalytic activity, recruit additional factors, or position the DH domain properly with respect to the GTPase by membrane targeting (26). A crystal structure of the Tiam1-DH/PH-Rac1 indicated that the PH domain provides only simple structural stabilization of the DH domain (46), leaving open the possibility of more complex functions. However, in the case of a Dbs-DH/PH-Cdc42 crystal structure, the Dbs PH domain participates directly in binding Cdc42, through a set of interactions involving the switch 2 region of the GTPase (39).
Several reports have demonstrated that the PH domains of DH/PH cassettes can interact with phosphorylated phosphatidylinositides and thereby potentially modify the membrane association or activity of their linked DH domains (28, 43). There are a number of examples of in vitro GEF activity being regulated by interactions of PH domains with phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] or PI(3,4,5)P3. These include PH domains of Vav1, Dbl, Tiam1, Sos1, P-Rex, and SWAP-70 (13, 20, 24, 37, 40, 42, 45). In the case of Vav1 and Sos1, PI(3,4,5)P3 binding to the PH domain relieves an intramolecular interaction between the DH and PH domains, allowing Rac to access the catalytic DH domain surface (16). P-Rex is also substantially activated by PI(3,4,5)P3, alone as well as synergistically with Gß
s; however, whether this mechanism involves a disruption of an interaction between the PH and DH domains is not known (45). The situation with Tiam1 is less clear, with evidence existing both for and against allosteric activation of GEF activity by PI(4,5)P2 (13, 20), and further in vitro studies indicate that the PH domain of Tiam1 may be specific for PI(3)P (43).
To date, no systematic study has been undertaken to correlate the in vitro biochemical functions (e.g., phospholipid binding and GEF activity) of DH and PH domains with in vivo biological activity in a model organism. In this regard, we have previously described the unc-73 gene from Caenorhabditis elegans (44), which encodes multiple isoforms, including an extended polypeptide (UNC-73A) with two DH/PH cassettes and a shorter N-terminal protein with only one DH/PH cassette (UNC-73B). UNC-73B has an N-terminal Sec-14 homology region followed by a spectrin repeat region, the DH and PH domains, and an SH3 domain. Mutations in unc-73, which affect the UNC-73B isoform, cause a variety of defects in axon guidance and cell motility (15). The UNC-73B DH/PH cassette specifically activates mammalian Rac1 in vitro and can stimulate actin polymerization in Rat2 fibroblasts. Indeed, the ability of unc-73 to mediate axon guidance and actin polymerization is impaired by alteration of Ser 1216 in the first DH domain, which inhibits Rac GEF activity. UNC-73B is highly related to the Trio and Kalirin family of proteins present in vertebrates (3, 17) and Drosophila (4, 5, 30, 36), and observations pertaining to UNC-73B are likely to have general implications for metazoan animals. We have, therefore, used a combination of in vitro biochemistry and functional analysis in C. elegans to examine the functional role of the PH domain of UNC-73B in Rac activation and axonal pathfinding in vivo.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The baculovirus for wild-type and mutant glutathione S-transferase (GST)/UNC-73B DH/PH/SH3 were generated by using the PharMingen Baculogold system and expressed in monolayer Sf9 cultures. The mutations created for this study are Trp1502 to Ala (the W1502A construct), Lys1424/Arg1426 to Ala/Ala (KR to AA), Lys 1420/Arg1422 to Glu/Glu (KR to EE), and 
PH, which is the removal of residues 1398 to 1559. Mutations were created by using the Stratagene Quikchange site-directed mutagenesis kit. Mutations were carried out on wild-type cDNA (residues 1179 to 1642) present in pSL301 and subcloned into the EcoRI site of pAcGHLT-A. These UNC-73B proteins were purified as described previously (44). For mammalian expression, the wild-type and mutated sequences were amplified by PCR and subcloned into the pEGFP-C1 vector from Clontech via the BamHI site. Further details are available upon request.
The minigene construct for worm microinjections containing the wild-type sequence and C-terminal Flag3 construct was made by mutating by PCR the stop codon of the pSL301-DH/PH/SH3 construct into a BamHI site and subcloning the mutated NruI/BamHI fragment into the previously described UNC-73B minigene (44). Subsequently, an annealed pair of Flag3-expressing oligonucleotides was subcloned in frame at the BamHI/NotI sites on this minigene, and the 3' untranslated region of UNC-73B was amplified by PCR and reintroduced into the NotI site of the minigene. Mutations were transferred into the minigene by digestion with NruI and BamHI from the pEGFP-C1 constructs. All mutations and subcloned portions of the cDNA were verified by DNA sequencing.
The pGex-CeRAC1, CeRHO, and CeCDC42 constructs were initially characterized and supplied by Louis Lim (8, 9, 10). The pGex-MIG-2 construct was a gift from Cynthia Kenyon (47). Purification of these GTPases was carried out as previously described (44).
Nucleotide exchange assays.
A fluorescence assay in which the C. elegans GTPases were preloaded with 12.5 µM mant-GDP (Molecular Probes) by incubation with the fluorescently labeled nucleotide in the presence of 6.25 mM EDTA for 30 min at room temperature was used (1). The loading was then quenched by adding MgCl2 to a final concentration of 50 mM; unbound nucleotide was removed, and protein was transferred to a buffer containing 20 mM HEPES (pH 7), 50 mM NaCl, and 2 mM MgCl2 by using a NAP-10 gel filtration column. For each reaction, 0.5 µM GTPase and 0.2 µM GEF were added together with 2.5 µM GTP in a volume of 800 µl. The time course of fluorescence was monitored with an LS-50B spectrophotometer (Perkin-Elmer) with 
excitation at 350 nm, emission at 440 nm (slits 5/15 nm), and an emission filter in place at 390 nm.
To test for the effect of phospholipids on GEF activity, various water-soluble diethyl-phosphorylated phosphatidylinositides (Echelon) were added prior to the beginning of each reaction at a final concentration of 25 µM with respect to phosphatidylinositide concentration.
Phospholipid dot blot. PIP strips were obtained from Echelon, and dot blots were carried out according to the manufacturer's suggested protocol with a few modifications. Strips were blocked overnight at 4°C in Tris-buffered saline containing 10% glycerol (TBSG) and 3% bovine serum albumin. Baculovirus-produced GST/UNC-73 DH/PH/SH3 wild-type and mutant fusion proteins were purified with glutathione-Sepharose (Pharmacia) as before and then washed in kinase buffer and labeled with [32P]ATP by using the catalytic subunit of protein kinase A (Sigma) as suggested by the manufacturer's protocol. The 32P-labeled fusion proteins were then washed with phosphate-buffered saline, eluted once with 10 mM glutathione in phosphate-buffered saline, and then applied to the PIP strips and incubated at a final concentration of 1 µM in 5 ml 3% BSA-TBSG for 50 min at room temperature. Each blot was then washed five times for 10 min each in TBSG and developed with a phosphorimager.
C. elegans strains and manipulations.
The unc-73 mutant allele e936 has been previously described (44). Transformation was performed as described previously (35) by coinjecting the UNC-73B minigenes with the plasmid pAC12 (20 ng/µl), which contains the motorneuron-specific promoter region of UNC-129 fused to GFP (11). This allowed us to assay the circumferential navigation of the DA and DB neurons, which migrate dorsally from ventral cell bodies to form the dorsal nerve cord, into e936/dyp5 balanced heterozygotes and maintained as extrachromosomal arrays. Homozygous e936 worms carrying the various arrays were then collected. Four different concentrations of the UNC-73B minigene (40, 10, 5, and 1 ng/µl) were tested for rescue. The ability of the UNC-73B wild-type and KR-to-AA minigenes and the inability of the KR-to-EE, W1502A, and PH minigenes to rescue the unc-73(e936) worms occurred consistently over the range of concentrations tested. Animals injected at 5 ng/µl were counted for axon guidance defects. At all concentrations of the W1502A minigene, homozygous e936 animals were sterile, preventing maintenance of that line in the mutant worms. Thus, in this case, homozygous e936 animals from heterozygous e936/dpy5 worms carrying the W1502A array were counted. Although there is the possibility of maternal contribution of UNC-73 to these animals, this contribution in itself is not enough to rescue the axon guidance defects in these worms. Living animals were mounted on a slide in a small drop of 10-mg/ml levamisole on a 2% agarose pad for microscopy. Axons that exhibited more than a single morphology were scored as containing the more severe defect (i.e., in descending severity, outgrowth defect > longitudinal defect > branching defect > oblique migration > normal migration) (22, 38).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The UNC-73B PH domain interacts with phosphorylated phosphatidylinositides.
To investigate the role of the PH domain in UNC-73B function, we generated four mutants with changes in the PH domain-coding sequence. To evaluate the possible effects of phosphatidylinositide binding (see below), we altered positively charged amino acids located in the ß1/ß2 loop of the UNC-73B PH domain, which correspond to residues involved in phosphatidylinositide binding in other PH domains. Based on the sequence alignment of the UNC-73B PH domain with the Dbs, Sos1, and Tiam1 PH domains, Lys1424 and Arg1426 are the most conserved basic residues in these proteins and were, therefore, initially targeted for simultaneous replacement by Ala (Fig. 2). However, since a double mutation of both residues to alanine (hereafter referred to as KR to AA) did not abolish phospholipid binding (see below), a second double mutant was made, changing both Lys1420 and Arg1422 to negatively charged Glu residues (referred to as KR to EE) (Fig. 2). Also, another pair of mutations was created to investigate other potential roles for the PH domain, such as direct or indirect involvement in Rac binding and activation, as well as a potential role in other protein-protein interactions. One mutation changed the conserved Trp1502 to Ala (W1502A) in the C-terminal 
-helix portion of the PH domain, which was expected to have a global effect on PH domain folding and function. Finally, a truncated UNC-73B protein in which the entire PH domain was deleted was created.
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As shown in Fig. 3, the DH/PH/SH3 fragment of UNC-73B recognized several phospholipids, binding preferentially to the monophosphorylated phosphatidylinositides [PI(3)P, PI(4)P, and PI(5)P], more weakly to the bisphosphorylated lipids tested, and poorly to PI(3,4,5)P3. The PKB/Akt PH domain, as a positive control, demonstrated strong and specific interaction with PI(3,4)P2 and PI(3,4,5)P3. GST protein alone did not display binding to any phospholipid (data not shown). This profile of interaction for UNC-73B is very similar to that seen for the PH domains of intersectin and Dbs (43).
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PH construct (GST/UNC-73B DH/SH3) showed no binding to any phospholipid, confirming that the PH domain of UNC-73B is responsible for its interaction with phosphorylated phosphatidylinositides. The KR-to-EE mutation also abolished the PH domain interaction with phospholipids, while the KR-to-AA mutation, in contrast, was still able to bind with a profile similar to that of the wild type (Fig. 3). The KR-to-EE mutation therefore had a more deleterious effect on binding the negatively charged phospholipids than did the KR-to-AA mutation, and this allowed us to investigate the effects of altering PH domain-phospholipid binding on the in vivo function of UNC-73B.
Surprisingly, the W1502A mutant was also able to interact with phospholipids. This substitution, based on structural studies of other PH domains, might be expected to destabilize the PH domain. PH domain mutants tested previously for in vitro binding to phospholipids have focused on the ß1/ß2 and ß3/ß4 loop regions of the PH domain, which were identified as the binding face for phospholipid interaction. Based on the previous work describing the Dbs-DH/PH-Cdc42 structure (39), the C-terminal -helix of the PH domain containing the invariant Trp and the phospholipid binding sites are on the opposing sides of the domain, and it is conceivable that replacement of the invariant Trp with Ala spares phospholipid binding. Our results show that the W1502A mutant retains an ability to interact with acidic phospholipids, which suggests that (at least for the UNC-73B PH domain) the resulting destabilization of the PH domain structure is not sufficient to prevent lipid binding.
Alteration of the UNC-73B PH domain affects in vitro GEF activity of the DH domain. Structural studies with Dbs and Tiam1 in complexes with Cdc42 and Rac1, respectively, show a role for their PH domains in DH domain stability and, in the case of Dbs, direct contact of the PH domain with the GTPase (39, 46). We therefore investigated the consequences of the UNC-73B PH domain mutations on the catalytic efficiency of the neighboring DH domain and on actin polymerization in a cell-based assay.
To quantify the effect of the PH domain mutations on the catalytic efficiency of the DH GEF activity, we used the fluorescent GEF assay. As a negative control, we used a protein with the S1216F substitution in the UNC-73 DH domain, induced by the unc-73(rh40) mutation, which causes severe axon guidance and cell migration defects due to its disruption of the integrity of the DH domain. This mutant failed to release mant-GDP from bacterially expressed, preloaded CeRAC1 (Fig. 4A) (44). We also found that the W1502A PH mutation adversely affected the activity of the DH domain (Fig. 4A), resulting in a loss of GEF activity very similar to that observed with the S1216F mutation. Likewise, the PH mutant also demonstrated a large reduction in GEF activity. These results, taken together with microinjection data (see below), show that the PH domain plays a positive role in activation of CeRAC by the UNC-73B DH domain.
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PH domain construct consistently expressed at much higher levels than the wild-type protein.
As reported previously, nuclear microinjection of Rat2 cells with an expression construct encoding the wild-type UNC-73B DH/PH/SH3 fused to GFP induced high levels of polymerized actin at the plasma membrane (44). This is consistent with the ability of the UNC-73B DH domain to activate Rac, which in turn induces actin polymerization (Fig. 5A, panel i). The cellular activity was dependent on the PH domain, as the PH construct failed to produce lamellipodia above basal levels (Fig. 5A, panel ii). Likewise, the W1502A PH mutant was also inactive in this assay. In contrast, mutants with substitutions in the ß1/ß2 loop of the PH domain retained an ability to induce actin polymerization (Fig. 5A, panels iv and v). Thus, although the PH domain is required for GEF activity, abolishing the phospholipid binding properties of the PH domain (KR-to-EE mutant) did not adversely alter actin-polymerizing activity when the mutant protein was overexpressed in Rat2 cells. This is potentially due to the high level of expression from microinjection of the plasmid into Rat2 cells.
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To determine whether the PH domain was necessary for this colocalization in Rat2 fibroblasts, we determined the localization of the PH and W1502A proteins in fibroblasts containing an activated form of Rac. To do this, we coinjected these two constructs with the constitutively active Flag V12-Rac construct and determined their localization by staining for anti-GFP. Figure 5B shows an overlapping of staining of PH and W1502A with the polymerized actin, indicating that colocalization of UNC-73B with lamellipodia occurs independent of the PH domain, potentially through its SH3 domain and/or DH domain.
GEF activity of UNC-73B DH/PH/SH3 is not affected by phospholipids. The ability of the UNC-73B PH domain to associate with phosphatidylinositides, coupled with previous reports of phosphatidylinositide-induced modulation of GEF catalysis in solution (13, 16, 24, 45), led us to evaluate the effects of phosphatidylinositides on UNC-73B-catalyzed GEF activity. To avoid contaminating the reaction with organic solvents required to solubilize long-chain phospholipids, which has been shown to inhibit Dbs GEF activity (43), we used water-soluble diethyl analogs of phosphatidylinositides as used for the investigation of Vav and Sos dependence of phospholipids on activity (16). The addition of a 25 µM concentration of either diethyl-PI, diethyl-PI(4)P, diethyl-PI(4,5)P2 or diethyl-PI(3,4,5)P3 did not significantly affect catalysis by UNC-73B DH/PH/SH3 on the preloaded mant-GDP/CeRAC complex (Fig. 6). This result corresponds to previous observations that phosphatidylinositides failed to alter nucleotide exchange by intersectin, Dbs, and Tiam-1 on mammalian Rac1 or Cdc42 (43). The effect of the water-soluble lipids on the PH domain mutants of UNC-73B was also tested, and no significant change in activity for any of the mutants in the presence or absence of these lipids was detected (data not shown). Therefore, unlike Vav and Sos1, the UNC-73B GEF activity is not obviously altered by soluble phosphorylated phosphatidylinositides, and the DH domain catalytic function is not apparently influenced by an allosteric mechanism involving PH domain inhibition by phosphatidylinositides.
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The unc-129::gfp marker was injected alone to score for the degree of misguidance that occurs in unc-73(e936) animals. These animals have an inactive Unc phenotype, are small and dumpyish, and demonstrate axon guidance defects. As shown in Fig. 8, a majority of motorneuron axons are severely misguided, with 64% of them exhibiting either a branching morphology, a longitudinal trajectory, or outgrowth defects. Establishment of a stable extrachromosomal array in unc-73 (e936) animals carrying the wild-type UNC-73B minigene rescued the Unc phenotype of these animals. The wild-type extrachromosomal transgene array also caused a reduction in the fraction of severely misguided axons to 22.5%.
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In contrast, the transgene array encoding the KR-to-EE mutation was not able to rescue the Unc phenotype of the e936 homozygous animals, even though this mutant can stimulate GEF catalysis in the context of the DH/PH cassette, both in GEF assays on modified and unmodified CeRAC and by microinjection into Rat2 fibroblasts (Fig. 8). This result suggests the importance of the positive electrostatic surface of UNC-73B's PH domain, a characteristic of a large number of PH domains, for UNC-73B biological activity. Since the KR-to-EE mutant can still enhance GEF activity in vitro, these data also indicate that the UNC-73B PH domain has an additional function in vivo that is important for activation of CeRAC and axon guidance, which correlates with its phospholipid-binding ability.
The minigenes containing the PH deletion and the W1502A point mutation were also unable to rescue axon guidance in the unc-73(e936) mutant, despite the ability of the W1502A construct to interact with phosphorylated phosphatidylinositides (Fig. 8). This is consistent with the finding that proteins carrying these PH domain mutations were inactive in UNC-73B DH domain-directed GEF catalysis in vitro.
Previous work has shown that both a dominant active form of mig-2 and loss of mig-2 function result in similar axon guidance defects in C. elegans (47); in addition, mutations in the PH domain of Vav have been reported to give dominant active mutants (34). It was, therefore, important to determine whether the UNC-73B mutants that failed to rescue axon guidance in vivo exert a dominant active effect. To distinguish between dominant negative and dominant positive mutations, we injected the unc-73 minigenes into e936/dpy5 heterozygous worms, which contain the wild type unc-73 allele tightly linked to the mutant dpy5 allele and which are phenotypically wild type. Thus, expression from a potential dominant active transgene plus the contribution from the wild-type allele would result in worms that exhibit a more severe Unc phenotype than they would have in the e936 homozygous state.
By this assay, none of the transgenes that failed to rescue the unc-73 defect were dominant active mutations. Rather, they appeared to have a dominant negative effect, as indicated in Fig. 8, since the KR-to-EE, W1502A, and PH transgenes gave a more severe phenotype in vivo with respect to axon guidance than GFP alone in the e936 homozygous background. This is potentially explained by the fact that the e936 allele is a mutation of the splice donor site of intron 16, which results in severe reduction, but not complete removal, of wild-type UNC-73B protein (44). Thus, the mutant transgenes may interfere with the function of the low levels of endogenous wild-type UNC-73B produced in the unc-73(e936) homozygous mutant background.
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DISCUSSION |
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With respect to GEF catalytic activity, deleting the UNC-73B PH domain results in a marked decrease in DH domain function in vitro, as noted previously with Trio (32). However, we have also found that mutating the highly conserved Trp1502 to alanine in the PH domain yields a mutant UNC-73B protein that is still capable of binding phosphorylated phosphatidylinositides but lacks GEF activity. Furthermore, a W1502A mutant UNC-73B minigene could not rescue the axon guidance defects of worms with a loss-of-function mutation in the endogenous unc-73 gene. These data indicate that the PH domain may have a direct effect on GEF activity that is important for in vivo function.
The PH domain could aid in GEF activity by stabilizing the DH domain structure, by direct binding to CeRAC, or both. Of interest is that recent crystallographic data for Cdc42 in complex with Dbs demonstrates that the ß1 strand and the ß3/ß4 loop of the Dbs PH domain directly interact with switch 2 of the GTPase (39). Those authors noted that the PH domain residues involved in the GTPase interaction are conserved in a subset of Dbl family proteins, of which Trio and UNC-73B are members. The in vitro GEF activities of Dbs, UNC-73B, and the N-terminal Trio DH domains are all enhanced very significantly by the presence of their PH domains. Also, the nuclear magnetic resonance structure of the Trio N-terminal DH domain demonstrates that it is properly folded in the absence of its PH domain, adopting a structure very similar to that of other DH domains (32, 2). Since the DH domain structure does not seem to require the PH domain, a plausible explanation for the ability of the Trio, UNC-73B, and Dbs PH domains to accelerate in vitro GEF activity is that the PH domain makes direct contact with the GTPase and assists in catalysis. It is apparent that some GEFs (Vav, for example) can function efficiently in the absence of their PH domains, but this ability is not universally shared by all DH/PH-containing proteins. Our results provide functional in vitro and in vivo data in support of the view, suggested by the Dbs-DH/PH-Cdc42 crystal structure, that the PH domain of a subset of DH/PH proteins contributes to exchange activity in a fashion that is physiologically relevant.
A well-recognized role of PH domains in general is to target proteins to specific sites in cellular membranes by interacting with phosphorylated phosphatidylinositides (29). The UNC-73B PH domain recognizes a number of phosphorylated phosphatidylinositides in vitro, although these data do not exclude the possibility that the UNC-73B PH domain has other binding partners. As in other PH domains, the ß1/ß2 loop is at least partly responsible for this interaction, since mutating two basic residues within this loop to glutamate abolished phospholipid binding. Phospholipid binding by the UNC-73B PH domain, however, is dispensable for Rac activation when ectopically overexpressed in cultured cells, since the KR-to-EE mutation (which disrupts phosphatidylinositide binding) caused actin accumulation at the plasma membrane in Rat2 fibroblasts and could promote the release of mant-GDP from CeRAC in vitro. However, the UNC-73B minigene containing the KR-to-EE mutation was incapable of rescuing unc-73 mutants in vivo, indicating that the binding of ligands such as phosphatidylinositides is important to the function of the UNC-73B PH domain motor axons in the intact worm. This may reflect the significant demands in correctly localizing a protein within the growth cone of an extending axon. A scheme consistent with our data is that UNC-73B can localize to the vicinity of the membrane through regions other than the PH domain, and this is followed by an interaction of the PH domain with phosphorylated phosphatidylinositides, allowing for the proper spatial organization of the DH/PH domain with the membrane-bound Rac (39) and resulting in the formation of a complex which is necessary for efficient CeRAC1/MIG-2 activation and correct axon guidance in vivo.
Although we do not know which phosphorylated phospholipid (or combination of phosphoinositides) is critical for in vivo activity of UNC-73B, there is a preference for the UNC-73B PH domain to bind monophosphorylated phosphoinositides in vitro. The lack of specificity that UNC-73B PH domain shows towards any single phosphatidylinositide is not unique and has been demonstrated with both the Dbs and intersectin PH domains. Most PH domains, including the UNC-73B PH domain, characteristically have a surface with a strong positive electrostatic potential that can mediate the interaction with the negatively charged phospholipids (7). The importance of this charged surface is supported by our data for UNC-73B. We also found no evidence for an allosteric enhancement of UNC-73B GEF activity by phosphorylated phosphatidylinositides in vitro. It is clear that PH domains in a number of GEFs exhibit different phospholipid specificities, with the advantage that this would allow for the unique temporal and spatial regulation of different GEFs and thus for different developmental processes to be regulated by the Rac GTPases (21). Indeed, activated Rac has been shown to interact with and activate type 1 PI(4)P 5 kinase (14), leading us to speculate that UNC-73B could be involved in formation of both PI(4,5)P2 and PI(3,4,5)P3, causing the subsequent activation of other GEFs during the signaling cascade involved in axon guidance, growth, and cell motility.
The UNC-73B PH domain may potentially be involved in intramolecular or intermolecular protein interactions necessary to target UNC-73B to the plasma membrane to allow it to carry out catalysis. Trio has been shown to interact with the actin binding protein filamin through its PH domain (6). We were able to reproduce this interaction of Trio with filamin in vitro (data not shown); however, attempts to demonstrate an interaction with UNC-73B and human filamin as well as a C. elegans form of filamin proved to be unsuccessful.
In conclusion, with the combined use of biochemical techniques and C. elegans as a model organism, we identified two functions of the UNC-73B PH domain that appear to be important in axon guidance in vivo. First, the PH domain has a direct effect on the ability of the DH domain to activate Rac GTPases. We infer that the UNC-73B PH domain is required to provide residues that facilitate binding to CeRAC/MIG-2 and/or maintain the structural integrity of the accompanying DH domain and thus to generate GTP-bound CeRAC/MIG-2 in the growth cones of specific neurons. Second, the positively charged face of the PH domain is important for binding to phosphoinositides and for axonal pathfinding, although phospholipid binding does not appear to allosterically affect DH domain GEF activity. The latter interaction may help to locate UNC-73B to specific sites in the extending axonal membrane where Rac activation is regulated by guidance cues. It will be of interest to determine how signals from guidance receptors regulate UNC-73B catalytic function in neurons.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the National Cancer Institute of Canada. J.C. was supported by a grant (NS41397) from the U.S. National Institutes of Health. T.P. is a distinguished investigator of the CIHR.
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FOOTNOTES |
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), MIG-2 (
), CeRHO (
), and CeCDC42 (•) or of GST to preloaded CeRAC (
) and MIG-2 (
) is shown. Proper loading of mant-GDP to CeRHO and CeCDC42 was demonstrated by monitoring release of the fluorescent nucleotide by EDTA (data not shown).
