The Lambeth laboratory studies some functions of the small GTPase Rac. Rac is a member of the Rho family of small GTPases. These proteins are isoprenylated at their C-termini, and the lipid is presumed to mediate protein-membrane interactions. The Rho family is a sub-family within the Ras superfamily. Rho family members regulate the cytoskeleton, cell growth and transcription. There are two closely related isoforms of Rac (Rac1 and Rac2) which differ in primarily in their C-termini, where Rac1 contains polybasic amino acid residues while Rac2 is less basic. Rac has been implicated in regulation of the cytoskeleton, the mitogenic response, superoxide generation in both phagocytes and transformed cells, and in regulation of transcription. Its known direct targets include the NADPH oxidase and PAK (p21-Activated Kinase).
Insert Region | Effector Region | Membrane Interactions
The structure of Rac1 determined by X-ray crystallography reveals that the effector region and the insert region each form distinct surfaces on Rac.The nucloetide is shown in violet, with light violet showing the positions of the three phosphates in GppNHp. The p67phox interaction surface contains the effector region (shown in redish brown), which is homologous to a region on Ras which undergoes a guanine nucleotide regulated change in conformation. In Ras, this "Switch I" region modulates interaction with the Ras target Raf1. The insert region (green) provides an additional surface for protein-protein interactions, but does not participate in the binding to p67phox. Approaches are being developed to investigate the direct interaction of Rac1 with other oxidase components. Also indicated is residue 181 of Rac1. The remainder of the C-terminus (residues 182-189) is not visible in the X-ray structure. This portion of the molecule is expected to help tether the Rac to the membrane.
Hirshberg, M., Stockley, R.W., Dodson, G., and Webbe, M.R. (1996) "The Crystal Structure of Human Rac1, a Member of the Rho-Family Complexed with a GTP Analogue" Nature Structural Biology 4, 147-152.
- Effector Region. The NADPH oxidase provides one of the best characterized examples of a signalling complex involving a small GTPase. The small GTPase Rac assembles with the cytosolic p47-phox and p67-phox and the membrane-associated flavocytochrome b558 to form the multi-component respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras decreases or eliminates the ability of Rac to support superoxide generation, and these mutations produce an elevated EC50 for Rac. These same mutations also interfere with the ability of Rac1 to activate PAK, and also interfere with cytoskeletal regulation and activation of the mitogenic response.
Diekmann, D., Abo, A., Johnson, C., Segal, A., and Hall, A. (1994) "Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity" Science 265, 531-533.
Freeman, J.L.R., Kreck, M.L., Uhlinger, D.J., and Lambeth, J.D. (1994) "Ras effector-homologue region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex" Biochemistry 33, 13431-13435.
Xu, X., Barry, D., Settleman, J., Schwartz, M., and Bokoch, G. (1994) "Differing structural requirements for GTPase-activating protein responsiveness and NADPH oxidase activation by Rac" J. Biol. Chem. 269, 23569-23576.
Kwong, C.H., Adams, A.G., and Leto, T.L. (1995) "Characterization of the effector-specifying domain of Rac involved in NADPH oxidase activation" J. Biol. Chem. 270, 19868-19872.
- Insert Region. We have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12 amino acid insert region (residues 124 -135) that has no counterpart in Ras (hence the name). Point mutations in and deletion of this region interfere with activation of the NADPH oxidase, but not PAK. These mutations cause an elevation in the EC50 for Rac in supporting superoxide generation. In addition, in vivo cytoskeletal regulation by Rac mutants is normal, but the mitogenic action of Rac1 is blocked.
Freeman, J.L., Abo, A., and Lambeth, J.D. (1996) "Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65" J. Biol. Chem. 271, 19794-19801.
- Membrane Interactions. The C-terminus participates in the binding of Rac to the membrane. Both isoprenylated Rac1 and isoprenylated Rac2 (both expressed in insect cells) can activate superoxide generation in cell-free systems. For non-isoprenylated forms expressed in E. coli, only Rac1 is effective. This is due to the ability of the polybasic region on Rac1 to interact with negatively charged residues on the membrane surface. Our data do not support the idea that this region is involved in specific protein-protein interactions in the assembled NADPH oxidase.
Kreck, M.L., Uhlinger, D.J., Tyagi, S.R., Inge,K.L. and Lambeth,J.D. (1993) "Participation of the small molecular weight GTP binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase: Inhibition by a C-terminal Rac peptide" J. Biol. Chem. 269, 4161-4168.
Kreck, M.L., Freeman, J.L., Abo, A. and Lambeth, J.D. (1996) "Membrane association of Rac is required for high activity of the respiratory burst oxidase" Biochemistry 35, 15683-15692.
|TABLE OF MUTATIONS|
|Rac||Activity||Binding to p67phox||Study|
|Rac1(N26H)||+||weak||(1) and present study|
|Rac1(D38N)||+||weak||(1) and present study|
|Rac1(M45T)||+||weak||(1) and present study|
|Rac1(Q61H)Ý||++||n.d.||Freeman and Lambeth, unpublished|
|Rac1(Y64F)||+++||n.d.||Freeman and Lambeth, unpublished|
|Rac1(D65N)||+++||n.d.||Freeman and Lambeth, unpublished|
|Rac1(V85E)||+++||n.d.||Freeman and Lambeth, unpublished|
|Rac1(R102E)||++++||n.d.||Kreck and Lambeth, unpublished|
|Rac1(H104A)||++++||n.d.||Kreck and Lambeth, unpublished|
|Rac1(H105A)||++++||n.d.||Kreck and Lambeth, unpublished|
|Rac1(K132E)||+||normal||(4) and present study|
|Rac1(L134R)||+||normal||(4) and present study|
Mutant proteins based on Rac2 were expressed in insect cells and were presumably at least partially isoprenylated, whereas all other mutant proteins were expressed in E. coli.
Ý Inhibits GTPase activity.
n.d., not determined.
1. Freeman, J.L.R., Kreck, M.L., Uhlinger, D.J., and Lambeth, J.D. (1994) Biochemistry. 33, 13431-13435.
2. Xu, X., Barry, D., Settleman, J., Schwartz, M., and Bokoch, G. (1994) J. Biol. Chem. 269, 23569-23576.
3. Diekmann, D., Abo, A., Johnson, C., Segal, A., and Hall, A. (1994) Science. 265, 531-533.
4. Freeman, J.L., Abo, A., and Lambeth, J.D. (1996) J. Biol. Chem. 271, 19794-19801.
5. Dorseuil, O., Reibel, L., Bokoch, G., Camonis, J., and Gacon, G. (1996) J. Biol. Chem. 271, 83-88.
6. Kwong, C.H., Adams, A.G., and Leto, T.L. (1995) J. Biol. Chem. 270, 19868-19872.
^ Up to beginning of Table ^
Mutational studies imply that Rac interacts directly with one or more components of the NADPH oxidase. Kinetic studies suggest that binding is concerted, and may involve multiple relatively weak interactions with more than one oxidase component. While strong interactions can frequently be observed for signalling proteins using overlay blotting or pull-down approaches, weak interactions are much more difficult to demonstrate. We have been able to show direct binding of Rac1 and Rac2 to p67phox was shown using a fluorescent analog of GTP [N-methylanthraniloyl(mant)GppNHp] as a reporter group. This analog binds tightly to Rac, and the fluorescent group shows an increase in fluorescence when p67 is added, indicating complex formation. Rac1 and Rac2 bound to p67phox with a 1:1 stoichiometry and with Kd values of 120 nM and 60 nM, respectively. Rac1 mutated in the effector region showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affects activity by inhibiting Rac binding to p67phox. Insert region mutations [Rac1(K132E) and L134R], while showing markedly elevated EC50 values, bound with normal affinity to p67phox. Thus, the effector region specifically interacts with p67phox, while the insert region interacts with some other NADPH oxidase component, possibly the cytochrome itself.
Nisimoto, Y., Freeman, J.F.R., Motalebi, S.A., Hirshberg, M. and Lambeth, J.D. (1997) "Rac binding to p67phox: Structural Basis for Ineractions of the Rac1 Effector Region and Insert Region with Components of the Respiratory Burst Oxidase" J. Biol. Chem. 271, in press.