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Structural requirements for PAK activation by Rac GTPases
U G Knaus1, Y Wang, A M Reilly
1Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA. uknaus@scripps.edu
Abstract:
The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.
Insights
Differences in the C-terminal polybasic domains of Rac1 and Rac2 explain their distinct abilities to activate p21-activated kinase (PAK). This suggests Rac
Area of Science:
- Molecular and Cellular Biology
- Signal Transduction
- Protein-Protein Interactions
Background:
- Rho family GTPases, including Rac1 and Rac2, are crucial regulators of cellular processes like cytoskeletal dynamics and cell cycle progression.
- p21-activated kinases (PAKs) are key downstream effectors implicated in mediating cellular functions regulated by Rac and Cdc42 GTPases.
- Understanding the specific interactions between Rac isoforms and PAK is essential for deciphering their distinct roles in normal and disease states.
Purpose of the Study:
- To elucidate the structural basis for differential activation of PAK1 by Rac1 and Rac2.
- To identify specific domains within Rac and PAK proteins critical for their binding and kinase activity regulation.
- To investigate the role of C-terminal polybasic domains in Rac and basic regions in PAK in mediating effector interactions.
Main Methods:
- Utilized mutational analysis to probe the functional significance of specific amino acid residues in Rac and PAK.
- Assessed protein binding affinities between Rac isoforms and PAK1 using biochemical assays.
- Quantified PAK1 kinase activity in response to different Rac variants and mutants.
Main Results:
- Rac1 exhibited significantly higher binding affinity and kinase activation of PAK1 compared to Rac2.
- Mutations in the C-terminal polybasic domain of Rac1 drastically reduced PAK1 binding and activation, with domain swapping reversing these properties between Rac1 and Rac2.
- A basic region (Lys66-Lys67-Lys68) in PAK was identified as essential for efficient binding and activation by Rac and Cdc42, with specific mutations altering these interactions.
Conclusions:
- Differences in the C-terminal polybasic domains of Rac1 and Rac2 are the primary determinants of their distinct abilities to activate PAK.
- The polybasic domain of Rac represents a novel effector-binding interface, potentially enabling isoform-specific signaling.
- A basic region within PAK is critical for kinase activation, and mere binding of Rac/Cdc42 is insufficient for full activation.