Multiple Interactions of PRK1 with RhoA
FUNCTIONAL ASSIGNMENT OF THE HR1 REPEAT MOTIF*

Peter Flynn, Harry Mellor, Ruth PalmerDagger , George Panayotou§, and Peter J. Parker

From the Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom and the § Ludwig Institute for Cancer Research, University College London, Courtauld Building, 91 Riding House Street, London W1P 8BT, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

PRK1 (PKN) is a serine/threonine kinase that has been shown to be activated by RhoA (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650). Detailed analysis of the PRK1 region involved in RhoA binding has revealed that two homologous sequences within the HR1 domain (HR1a and HR1b) both bind to RhoA; the third repeat within this domain, HR1cPRK1, does not bind RhoA. The related HR1 motif is also found to confer RhoA binding activity to the only other fully cloned member of this kinase family, PRK2. Furthermore, the predictive value of this motif is established for an HR1a sequence derived from a Caenorhabditis elegans open reading frame encoding a protein kinase of unknown function. Interestingly, the HR1aPRK1 and HR1bPRK1 subdomains are shown to display a distinctive nucleotide dependence for RhoA binding. HRIaPRK1 is entirely GTP-dependent, while HR1bPRK1 binds both GTP- and GDP-bound forms of RhoA. This distinction indicates that there are two sites of contact between RhoA and PRK1, one contact through a region that is conformationally dependent upon the nucleotide-bound state of RhoA and one that is not. Analysis of binding to Rho/Rac chimera provides evidence for a HR1aPRK1 but not HR1bPRK1 interaction in the central third of Rho. Additionally, it is observed that the V14RhoA mutant binds HR1a but does not bind HR1b. This distinct binding behavior corroborates the conclusion that there are independent contacts on RhoA for the HR1aPRK1 and HR1bPRK1 motifs.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Guanine nucleotide-binding proteins provide a class of cellular switches that cycle between functional states defined by their GTP or GDP binding status. The specific roles of these GTP-binding proteins in the control of cell functions are many, ranging from the interaction of receptor coupled heterotrimeric G-proteins with second messenger generators (e.g. Ref. 2) to that of initiation control in translation (3). Among this broad class of proteins are the "small" GTP-binding proteins constituted by the Ras, Rho, Rab, Arf, and Ran families. These G-proteins have distinctive roles in cellular control, encompassing cell proliferation/differentiation (4), cytoskeleton organization (5), vesicle traffic (6), Golgi function (7), and nuclear transport (8). Targets for their action include the vesicle-associated protein rabphilin (9), a phospholipase D (10-17), and a number of protein kinases including protein-tyrosine kinases (e.g. Cdc42-Ack (18)) and protein serine/threonine kinases (e.g. Ras-Raf (19-21) and Rac/Cdc42-p65PAK (22, 23)).

Recently, a number of targets for Rho have been reported (1, 24-27). These have been identified through conventional protein-protein interactions as well as through yeast two-hybrid screening. Among the targets identified as Rho-binding proteins is the lipid-activated protein serine/threonine kinase PRK1/PKN1 (29-31). This kinase and two related sequences were originally identified in screens for protein kinase C gene products; however, it is clear that these proteins are in fact quite distinct from protein kinase C within their regulatory domains. The complete cDNA cloning of PRK1 and PRK2 has revealed conserved regulatory domain features in these predicted proteins absent from protein kinase C (31), although it should be noted that one domain is predicted to retain some structural resemblance to the protein kinase C C2 domain (32). PRK1 has been shown to bind to and be stimulated by RhoA in vitro (1); it has been demonstrated also to be activated by various fatty acids and phosphoinositides (33, 34). The precise molecular action of these effector molecules on PRK1 is poorly understood.

The potential importance of the Rho-PRK pathway in controlling cell functions has led us to define the site of interaction of these proteins. Interestingly, the results demonstrate that in fact RhoA can make two independent contacts with PRK1 that differ in their dependence upon RhoA conformation and lead to a high affinity interaction. The two target sites on PRK1 are homologous and define a class of binding sites for Rho.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

DNA Constructs-- The full-length PRK1 was inserted into the mammalian expression vector (Invitrogen). PRK1 cDNA was digested with BssHI and XhoI; the BssHI site was blunt-ended with mung bean nuclease, and the purified fragment was inserted into the EcoRV/XhoI sites of pcDNA3. The PRK2 HR1abc fragment (amino acids 1-380) was subcloned into the bacterial expression vector pGEX-2TK (Pharmacia Biotech Inc.) at the BamHI/EcoRI site. DNA fragments encoding various PRK1 HR1 motifs were amplified by PCR using Vent DNA polymerase (New England Biolabs) and the following oligonucleotide primers: HR1aPRK1, 5'-GGGATCCCCATGGCCAGCGACGCCGTG-3' and 5'-CCTCGAGTCAGGTGGCCGCCGGGTCGGG-3'; HR1bPRK1, 5'-GGGGATCCCCGCCACCAACCTGAGCCGC-3' and 5'-CCCTCGAGTCACGGGGCTGCCTGGTTCTC-3'; HR1cPRK1, 5'-GGGGATCCCCGATGACACCCAAGGGAGT-3' and 5'-CCCTCGAGTCAAGCGAGCTCTTCTCGCAG-3'; HR1abcPRK1, 5'-GGGATCCCCATGGCCAGCGACGCCGTG-3' and 5'-GGGGATCCCCGATGACACCCAAGGGAGT-3'; F46F6.2 HR1a, 5'-TGGGATCCCCGGCACTTATTGGGAGACA-3' and 5'-CCCTCGAGCTAAAAAGCTCCACTGTTGT-3'. The products were digested with BamHI and XhoI and subcloned into the BamHI/XhoI site of pGEX-5X-1 (Pharmacia). All of the constructs were confirmed by sequencing. The small G-protein constructs, both wild type and chimeric, have been described previously (35).

Purification and Nucleotide Loading of Recombinant Proteins-- The full-length PRK1 in pcDNA3 was transfected into COS7 cells by electroporation, expressed for 36 h, and harvested directly into SDS-PAGE2 sample buffer. The various cDNA constructs in pGEX vectors were expressed in the Escherichia coli Bl21 bacterial strain and purified by affinity chromatography on glutathione-Sepharose. The GST-GTPase fusion proteins were cleaved by thrombin treatment (3 units/liter of culture), whereas the purified kinase fragments were eluted as fusion proteins from the Sepharose with 10 mM reduced glutathione. The relative nucleotide loading capacities of the small GTPases used in all of the studies were determined by a guanine nucleotide filter binding assay (36). Briefly, GTPases were incubated with [alpha -32P]GTP in a low MgCl2 buffer for 20 min at 30 °C followed by the addition of excess MgCl2 and filtering through prewetted nitrocellulose using a Millipore filtration device. Filters were washed, and bound nucleotide was determined by scintillation counting. The relationship between mol of nucleotide retained on the filters and mol of GTPase present was used to calculate the percentage of nucleotide loading capacity.

Overlay Assay-- Proteins separated by SDS-PAGE were transferred to nitrocellulose, and the filters were incubated in renaturation solution: phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), 0.5 mM MgCl2, 50 µl of ZnCl2, 0.1% Triton X-100, and 5 mM dithiothreitol (25) for 15 h at 4 °C, followed by blocking in 1% BSA, 0.1% Tween 20 for 1 h. The recombinant, wild-type RhoA, which had previously been determined to have approximately 10% nucleotide loading capacity, was radiolabeled by incubating 10 µM protein with 20 µCi of [alpha -32P]GTP (3000 Ci/mmol) in PBS with 2 mM dithiothreitol, 5 mM EDTA, and 1 mg/ml BSA for 30 min at 30 °C. Nucleotide exchange was stopped by adding 10 mM MgCl2 and placing the reaction on ice. The filters were then incubated in PBS with 10 mM MgCl2 and 2 mg/ml of the radiolabeled RhoA for 30 min at 4 °C, followed by two washes in PBS containing 10 mM MgCl2 at 4 °C. The bound [alpha -32P]GTP-loaded RhoA was then visualized by autoradiography.

For the chimeric protein overlays, equimolar amounts of the HR1PRK1 constructs were subjected to SDS-PAGE and transferred to nitrocellulose followed by renaturation and blocking (as above). The nucleotide loading capacity of the Rho/Rac chimeras, as well as the positive and negative controls were determined prior to the overlay, which was carried out exactly as above.

BIAcore-- BIAcore methodologies employed were as previously published (37). All experiments were performed at 25 °C and at a constant flow of 5 µl/min in running buffer: phosphate-buffered saline, 10 mM MgCl2. For immobilization of proteins, a carboxymethylated dextran layer on a CM5 sensor chip (Pharmacia) was activated by a 40-µl injection of a 1:1 mixture of 11.5 mg/ml N-hydroxysuccinimide and 75 mg/ml N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide. Proteins were diluted in coupling buffer (10 mM acetate buffer, pH 4.5) and injected over the activated surface. Excess, uncoupled sites were blocked by an injection of 1 M ethanolamine. Rho proteins were passed through small desalting columns (NICK columns, Pharmacia) to exchange them into running buffer (as above) and were then injected over the immobilized proteins at a concentration of between 10 and 20 µg/ml. The response was recorded in arbitrary resonance units (RU), and the resulting RU versus time plots were analyzed with the evaluation software supplied with the instrument.

Solution Binding Assay-- Equimolar amounts of the PRK1 GST fusion proteins; HR1abcPRK1, HR1aPRK1, HR1bPRK1, HR1cPRK1, and GST control were bound to glutathione-Sepharose, which was packed into Clontech minicolumns (50-µl bed volume). Any nonspecific sites on either the column or the Sepharose were then blocked by washing the columns with Buffer A: PBS containing 10 mM MgCl2, 1 mg/ml BSA, and 0.1 mM GTP or GDP.

The recombinant wild type RhoA (10 or 20 µg, as indicated) was loaded with either GTP or GDP by incubation with an excess of the nucleotide in the presence of 2 mM dithiothreitol, 5 mM EDTA, and 1 mg/ml BSA at 30 °C for 30 min. The reaction was stopped by the addition of 10 mM MgCl2. The nucleotide-loaded RhoA was applied to the Sepharose columns, and the flow through the columns was blocked for an incubation period of 1 h. The columns were then washed with Buffer A using either 0.4 ml (8 × Sepharose bed volume) for the experiment shown in Fig. 5A or 0, 0.25, 1.00, and 4.00 ml for the experiment in Fig. 5B. Proteins were then eluted using 20 mM glutathione. Both the application of the RhoA and the subsequent washes were carried out at 4 °C, whereas the glutathione-dependent elution was performed at room temperature.

Samples of the eluate were subjected to 15% SDS-PAGE, transferred to nitrocellulose, and blotted for RhoA. The same procedure was employed for the Caenorhabditis elegans GST-HR1a immobilized on glutathione-Sepharose.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

PRK1 has been identified as a target for RhoA through yeast two-hybrid analysis and direct protein-protein interaction (see Introduction). To delineate the RhoA binding site on PRK1, attention was focused on the noncatalytic regulatory domain of the protein. We had previously identified two domains in PRK1, HR1 and HR2, based upon conservation with PRK2 (see Fig. 1). The full-length PRK1 as well as these two regulatory domains were expressed, and binding of [32P]GTP-bound RhoA was determined by overlay. Full-length PRK1 itself bound RhoA in this assay as shown in Fig. 2A. No equivalent binding activity was detected in COS cell extracts from vector-transfected cells. An E. coli-expressed GST fusion of HR1PRK1 also bound RhoA, while GST itself did not (Fig. 2B). No binding was observed for HR2PRK1 (data not shown). Consistent with the binding activity of the HR1PRK1 domain, the homologous domain from PRK2 was found also to bind RhoA (Fig. 2B). The specificity with respect to G-protein was tested by comparison with [32P]GTP-bound Rac1 and Cdc42, neither of which were found to bind HR1PRK1 effectively (data not shown; see below).


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Fig. 1.   General domain structure of the PRK protein family. A, shown are the catalytic domain; the HR1 domain, which is made up of a repeated subdomain; and the HR2 domain, which has been shown to have homology with the protein kinase C C2 domains. B, the HR1 domain (Rho binding domain; see "Results") has repeated motifs, which are conserved between members of this family of proteins. The individual repeats from the tripartite HR1abc are indicated for PRK1, PRK2, and the C. elegans open reading frame F46F6. Conserved hydrophobic sequences (open circle ) are highlighted in light shading. Similarly conserved acidic residues (E/D) are shown in black shading, and basic residues (+) are shown in dark shading. The conserved glycine (G) and asparagine (N) are boxed.


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Fig. 2.   RhoA binds to PRK1 and PRK2 via the HR1 repeat. Binding of RhoA [alpha -32P]GTP via ligand overlay assay was assessed for the following. A, full-length PRK1 (amino acids 1-942) transiently overexpressed in COS cells; whole cell lysates were fractionated by SDS-PAGE and transferred to nitrocellulose for the overlay assay. Vector-transfected cell extracts are shown as a control. B, HR1PRK1 (amino acids 1-293) was expressed as a GST fusion protein, HR1PRK2 (amino acids 1-380 was a His-ragged construct). The bacterial proteins were fractionated by SDS-PAGE and transferred to nitrocellulose for overlay (see "Experimental Procedures").

Since we had defined the HR1 domain as three consecutive repeats of a ~100-amino acid motif, these were expressed individually as subdomains, as defined under "Experimental Procedures." The a, b, and c repeat motifs (denoted HR1aPRK1, etc) were expressed as GST fusion proteins and purified for analysis by overlay. Interestingly, both the HR1aPRK1 and HR1bPRK1 GST fusion proteins bound RhoA; no binding was observed for HR1cPRK1 nor for the GST control (Fig. 3A). The greater sequence similarity between HR1aPRK1 and HR1bPRK1 compared with HR1cPRK1 (Fig. 1) is consistent with this pattern of behavior.


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Fig. 3.   HR1aPRK1 and HR1bPRK1 bind to RhoA. RhoA binding was assessed employing the ligand overlay assay for the following. A, the PRK1 HR1 repeated motifs, HR1aPRK1 (amino acids 1-106), HR1bPRK1 (122-199), and HR1cPRK1 (200-293) as defined under "Experimental Procedures." The proteins were produced as GST fusions and purified on glutathione-Sepharose. Binding is shown in the upper panel, and protein staining is shown in the lower panel. GST itself serves as a control. B, the C. elegans open reading frame F46F6 contains an HR1a domain, which was expressed as a GST fusion (amino acids 1-106). Binding (upper panel) and protein staining (lower panel) are shown.

The RhoA binding property ascribed to both the HR1aPRK1 and HR1bPRK1 repeats suggests that this defines a subdomain that has predictive value for RhoA targets. To test this notion, we identified a C. elegans open reading frame encoding a protein kinase that has an amino-terminal HR1 domain. The HR1a sequence from this gene was cloned, expressed as a GST fusion protein, and purified. This repeat was also found to bind RhoA in an overlay assay (Fig. 3B). The data indicate that the HR1a repeat provides a useful predictive sequence for the identification of Rho targets.

To further characterize the multiple HR1PRK1-RhoA binding sites and define the nucleotide dependence of this interaction, binding was monitored using a BIAcore; this permitted a real time evaluation of RhoA-HR1PRK1 interactions. For these studies, RhoA was loaded with nucleotide immediately prior to use. As illustrated in Fig. 4, the complete HR1PRK1 (HR1abc) domain bound RhoA in a GTP-dependent manner. Consistent with the overlay assay, both HR1aPRK1 and HR1bPRK1 but not HR1cPRK1 also bound RhoA. However, in contrast to HR1aPRK1, where binding was completely specific to GTP-RhoA, for HR1bPRK1 a degree of binding over background was observed for GDP-RhoA. There was no specific binding of RhoA (GTP- or GDP-loaded) to the HR1cPRK1 domain when compared with the GST control.


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Fig. 4.   HR1PRK1 and subdomain binding to RhoA. The five panels indicate the BIAcore 2000 (Pharmacia Biosensor) measurements of change in RU over time for binding of bacterially expressed RhoA (preloaded with either GTP or GDP) to immobilized GST fusion proteins: PRK1 HR1abcPRK1 and GST control (A) and HR1aPRK1, HR1bPRK1, andHR1cPRK1 (B), as indicated within each panel.

While it is possible to obtain kinetic parameters (such as association and dissociation rates) from BIAcore data, a simple correlation between the present data and defined kinetic models for a one- or two-site interaction was not seen. Furthermore, the immobilized ligands were found to be unstable after a few rounds of interaction and regeneration, precluding the determination of equilibrium binding over a range of RhoA concentrations. It should be noted also that the complexity of HR1-RhoA interactions does not reflect mass transport-related distortions as judged by plots of ln(abs dR/dt). Thus, the data indicate that the binding of HR1 and RhoA is not simple in this context. This situation might have arisen from the dimerization state of the immobilized fusion proteins due to their GST tags; however, the observed homooligomerization of the individual HR1 repeats after cleavage from their expression tags3 precluded the use of cleaved proteins as a means of obtaining a clearer result. Consequently, the comparisons made here are semiquantitative; proteins that give the highest response in RU are presumed to have the highest affinity for the immobilized ligand, i.e. HR1abcPRK1 > HR1aPRK1 > HR1bPRK1 >>  HR1cPRK1. This relative efficacy and the nucleotide dependence are consistent with the solution binding studies (see below).

To assess further the distinctive nucleotide dependence of HR1 subdomain-RhoA interactions noted on the BIAcore, direct binding and dissociation were monitored for GST-HR1PRK1 constructs immobilized on glutathione-Sepharose beads. Consistent with the binding data above, it was found that immobilized HR1aPRK1 and HR1bPRK1 bound RhoA, while HR1c PRK1 did not. The binding of Rho to the C. elegans HR1a domain was also tested. This HR1aF46F6.2 domain behaved exactly as HR1aPRK1 (see Fig. 6) and was found to display complete GTP dependence for maintenance of RhoA binding (Fig. 5B).


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Fig. 5.   HR1aPRK1 and HR1bPRK1 repeats display distinct nucleotide dependences for RhoA binding. A, immobilized HR1 constructs were assessed for binding to RhoA (20 µg) using a simple one-step washing procedure. Bound RhoA was detected by elution of the complex with glutathione and blotting for RhoA. B, binding to the C. elegans HR1a was assessed, as for panel A.

The dissociation of these RhoA complexes was assessed by a sequential washing protocol. The stable association of HR1abcPRK1 to RhoA occurred with a distinct, but not exclusive, preference for the GTP-bound state (Fig. 6). For HR1aPRK1, sequential washing revealed no stable binding of GDP-RhoA, while GTP-RhoA was retained. HR1bPRK1 was found to bind stably to GDP-RhoA in addition to GTP-RhoA, with equivalent dissociation after washing with 20 bed volumes (Fig. 6). The different properties of the HR1aPRK1 and HR1bPRK1 in the retention and dissociation of GTP-RhoA and GDP-RhoA are consistent with the BIAcore data. These results demonstrate that although homologous, these HR1 repeats make different contacts with RhoA. Furthermore, it appears that the specific properties of the HR1aPRK1 repeat are retained for HR1aF46F6.2.


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Fig. 6.   Sequential washing was employed to define the dissociation behavior of the HR1 repeats with respect to their nucleotide dependences for RhoA (10 µg) binding. Following various volumes of washing as indicated, the bound RhoA complex was eluted with glutathione and detected by Western analysis. Purified GST itself was employed as a control. Autoradiographs were analyzed for signal intensity to give arbitrary units for the amount of RhoA bound to Sepharose. black-square, RhoGTP; black-diamond , RhoGDP.

It is implicit in the conclusions above that HR1aPRK1 and HR1bPRK1 bind distinct sites on RhoA. To establish whether these are overlapping or whether in fact two simultaneous contacts might be made between RhoA and PRK1, "fragmentation" of RhoA was employed through the use of the Rac-Rho chimera. It has been established previously that PRK1 does not bind Rac (1); however, here a very low level of Rac association with HR1a is reproducibly seen (see below). This degree of binding is mirrored by the Rac143Rho chimera. The chimera were loaded with equal efficiency (see "Experimental Procedures"), demonstrating the integrity of each of the constructs employed. These chimera were assessed for interactions with HR1aPRK1, HR1bPRK1, and HR1cPRK1 using an overlay assay, to reduce any influence of the oligomeric state of the HR1 proteins on binding. As shown in Fig. 7A, it was observed that the Rac-(1-73)/Rho chimera bound only to HR1aPRK1, clearly distinguishing the sites of HR1aPRK1 and HR1bPRK1 binding. The Rac-(1-143)/Rho chimera bound neither HR1aPRK1 nor HR1bPRK1. Expression of the RhoA-(1-73)/Rac chimera did not yield sufficient material for analysis of HR1a/bPRK1 binding due to protein instability; this problem has been noted previously (35). These observations indicate a site of HR1aPRK1 interaction with RhoA between residues 73 and 143 and suggest that the HR1bPRK1 may interact with RhoA within the amino-terminal 73 amino acids.


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Fig. 7.   HR1aPRK1 and HR1bPRK1 bind to distinct regions of RhoA. A, overlay studies using Rho, Rac, and the indicated chimeric proteins are shown. The G-proteins were loaded with [alpha -32P]GTP and incubated with the immobilized HR1 repeats as indicated. Filters were washed and autoradiographed (see "Experimental Procedures"). Specific binding is observed for the HR1aPRK1 (RhoA and Rac73/RhoA) and HR1bPRK1 (RhoA) proteins only. B, an overlay with V14RhoA was carried out as indicated in the legend for panel A.

To further assess the binding of HR1b to the amino-terminal region of RhoA, the "activating" V14RhoA mutant was employed. As shown in Fig. 7B, the mutant failed to bind HR1b while retaining binding to HR1a. Since HR1b binds GTP-RhoA, the inability of this domain to bind V14RhoA implies a contact within the amino-terminal region close to residue 14 and not a consequence of the activating conformational changes induced by this mutation.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Previous studies have identified PRK1 (PKN) as a target for RhoA (1, 27) and indeed demonstrated a modest activation of this protein kinase by GTP-RhoA (1). The work described here defines a two-site interaction between RhoA and PRK1. Interestingly, the two regions of contact within the PRK1 protein are in the regulatory domain and are defined by the first two HR1 repeats, a and b. Of the three repeats present, these are the two with the most similar amino acid sequences (see Fig. 1). Despite the similarity of the HR1aPRK1 and HR1bPRK1 repeats, these proteins have differing specificities for RhoA; HR1aPRK1 binds to only the GTP-bound form of RhoA, and HR1bPRK1 binds to both GTP- and GDP-bound forms. This implies that within the context of PRK1 itself, HR1aPRK1 makes contact with a conformation determined by GTP binding, while HR1bPRK1 does not. Indeed, the properties of the intact HR1abcPRK1 domain are consistent with this, given its preferential but nonexclusive binding to the GTP-bound form of RhoA. It can be concluded that tight binding of GTP-RhoA for intact PRK1 is effected through the combined contacts of the HR1aPRK1 and HR1bPRK1 motifs. These interactions are summarized in Fig. 8 (see below). That HR1abcPRK1 does indeed bind more tightly than either HR1aPRK1 or HR1bPRK1 alone is evidenced by the slower dissociation of RhoA seen for the immobilized HR1 repeats. Precise dissociation rates based upon the BIAcore analysis could not be derived, since the binding data did not fit rigorously the binding models tested.


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Fig. 8.   A summary of the interaction of RhoA and PRK1. In the upper part of the figure, the binding behavior of the recombinant HR1 domain is illustrated. GDP-RhoA is shown to interact at the single HR1b motif within the recombinant protein. In the GTP-bound state, RhoA contacts both the HR1a and HR1b motifs. For the intact PRK1, binding through HR1a and HR1b is likely to contribute to an activation process through relief of HR1 basic amino acid (pseudosubstrate site) interactions with the catalytic domain.

The elucidation of the binding activities of these HR1 repeat motifs indicates that there is indeed a reason for defining these as functional units. Previously, these repeats had been defined only on the basis of conservation between the PRK1 and PRK2 gene products (31). The demonstration that the HR1 domain of PRK2 also confers GTP-RhoA binding is entirely consistent with this analysis. Furthermore, the predictive nature of the HR1 repeat motif has been established by demonstration that such a sequence from a C. elegans open reading frame will also bind GTP-RhoA. Similar HR1 repeat sequences are found in numerous other proteins including rhotekin, which interestingly has only the HR1a motif and has been shown to bind Rho in a GTP-dependent manner (26). During revision of this submission, it was reported that PRK2 binds to RhoA in a nucleotide-independent manner (38). This is compatible with the finding here that PRK1 also makes a nucleotide-independent contact. However, for the intact RhoA binding domain of PRK1, a relative GTP dependence is observed, demonstrating a significant contribution from the HR1a-RhoA contact; it would appear that in PRK2, the predicted nucleotide-independent contact with the HR1bPRK2 domain is dominant. It is also notable that PRK2 will bind Rac, albeit less effectively than RhoA (38); the very weak binding of HR1aPRK1 to Rac observed here indicates a similar property for PRK1.

It has been reported recently that RhoA binds PRK1 through an amino-terminal region that encompasses the HR1a repeat (27). This is consistent with the results here, albeit only a partial solution to the RhoA-PRK1 interaction as defined here. Additionally, Ono and colleagues (39) have provided evidence for a pseudosubstrate motif in the basic region of the HR1a repeat, and it is possible that sequestration of this region alongside the basic region also conserved in the HR1b repeat contributes to the RhoA-dependent activation of PRK1 (see Fig. 8).

A notable feature of the PRK1-RhoA interaction is that these homologous repeats within the HR1 domain bind to distinct target sites on RhoA. The RhoA/Rac chimera analysis is consistent with HR1bPRK1 interaction with the RhoA amino terminus (residues 1-73) and HR1aPRK1 interaction with RhoA between residues 73 and 143 (see Fig. 7). The RhoA/Rac chimera (i.e. RhoA1-73Rac) that would be predicted to bind HR1bPRK1 but not HR1aPRK1 degrades during purification; hence, it has not proved possible to confirm this prediction. The finding that the V14 point mutant in RhoA fails to bind HR1b while still binding HR1a corroborates the conclusion from the chimera analysis that the HR1b binds to the amino-terminal region of RhoA. Furthermore, the more avid binding of RhoA to HR1abc compared with HR1a or HR1b indicates that both RhoA sites are contacted upon interaction with the intact HR1 domain. It is noteworthy that the binding of HR1abc to GDP-RhoA resembles that of HR1b alone; i.e. a single lower affinity contact is made for GDP-RhoA. While it is concluded that the binding of HR1abc to GTP-RhoA involves both HR1a and HR1b contact, independent proof of this from competition analysis has not been possible, since recombinant HR1a and HR1b domains interact directly in vitro.3 Furthermore, overlapping oligopeptides based upon RhoA covering the entire predicted binding regions for HR1a and HR1b (residues 1-100) are neither able to interact directly nor able to compete for HR1a/HR1b binding to RhoA, indicating that the sites of interaction on RhoA are sensitive to conformational constraints.

The site of interaction on RhoA for HR1aPRK1 (amino acids 73-143) overlaps the Switch II region of RhoA, suggesting that this may be involved in the GTP-dependent contact. By contrast, while HR1bPRK1 is predicted to bind to the amino-terminal region of RhoA (amino acids 1-73), the nucleotide dependence of this interaction indicates that the Switch I region is unlikely to be involved. One can speculate that duplication of this HR1 motif has permitted the evolution of a high affinity (two contacts) RhoA binding domain. It is of interest that the interaction of Ras with c-Raf is also thought to operate through two sites (40-42). However, in this case the points of contact in the effector (i.e. Raf) are not themselves homologous. A clear precedent for asymmetric interactions of homologous proteins is afforded by the growth hormone receptor; here the two identical receptors contact distinct sites on growth hormone causing dimerization (43). In PRK1, there is no evidence for dimerization, and in fact the HR1abcPRK1 domain is likely to form a discrete folded domain, since HR1bPRK1 alone behaves as a dimer on gel filtration.4 Whether this behavior is consequent upon the predicted leucine zipper that is part of each HR1 repeat motif awaits further analysis.

The results described here establish the nature of the RhoA binding site on PRK1 and PRK2. Further, the dissection of this site on PRK1 demonstrates that the strong interaction between regulator (RhoA) and effector (PRK1) is driven by a bipartite interaction involving sequential repeats on PRK1 and distinct regions on RhoA. The actual molecular organization of this interaction will require direct structural analysis.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Alan Hall for the RhoA, Rac, and Cdc42 constructs.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: The Salk Institute, Molecular Biology and Virology Laboratory, P.O. Box 85800, San Diego, CA 92186-5800.

To whom correspondence should be addressed.

1 To avoid confusion with the earlier description of the nerve growth factor-activated kinase "PKN" (28), the nomenclature employed here will be PRK (protein kinase C-related kinase) throughout.

2 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RU, resonance units.

3 P. Flynn and P. J. Parker, unpublished observations.

4 P. Flynn, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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