Evidence for a Novel Mechanism of the PAK1 Interaction with the Rho-GTPases Cdc42 and Rac

P21-activated kinase 1 (PAK1) is activated by binding to GTP-bound Rho GTPases Cdc42 and Rac via its CRIB domain. Here, we provide evidence that S79 in the CRIB domain of PAK1 is not directly involved in this binding but is crucial for PAK1 activation. S79A mutation reduces the binding affinity of PAK1 for the GTPases and inhibits autophosphorylation and kinase activity of PAK1. Thus, this mutation abrogates the ability of PAK1 to induce changes in cell morphology and motility and to promote malignant transformation of prostate epithelial cells. We also show that growth of the prostate cancer cell line PC3 is inhibited by the treatment of a PAK1-inhibiting peptide comprising 19 amino acids centered on S79, but not by the PAK1 peptide containing the S79A mutation, and that this growth inhibition is correlated with reduced autophosphorylation activity of PAK1. Together, these findings demonstrate a significant role of S79 in PAK1 activation and provide evidence for a novel mechanism of the CRIB-mediated interaction of PAK1 with Cdc42 and Rac.


Introduction
PAK1 is a major downstream effector of the Rho-GTPases Cdc42 and Rac, which act as molecular switches that transduce various extracellular signals into intracellular responses [1]. PAK1, the best-characterized member of the PAK family, forms a transinhibited dimer in its inactive state, in which the catalytic domain of one PAK1 monomer is blocked by the autoinhibitory domain (AID) of the other [2,3]. This autoinhibitory conformation is disrupted by binding of the GTP-bound Cdc42 and Rac to the CRIB (Cdc42/Rac-interactive binding region) domain [4,5,6], leading to autophosphorylation at specific sites including T423 within the activation loop and consequent activation of PAK1 [7,8]. Efficient activation of PAK1 requires its membrane targeting. PAK1 is recruited to the plasma membrane via the SH3-containing proteins Nck and Grb2 [9,10,11], where it may be activated by signaling molecules such as PDK1 kinase [12], sphingosine [13,14] and PIP2 [15] in a manner independent of the GTPases.
PAK1 is frequently overexpressed and hyperactivated by dysregulation of a number of signaling pathways in human cancer cells that are stimulated by growth factor receptors such as EGFR, PDGFR, and VEGFR [16]. The activated PAK1 in turn promotes cancer cell invasion and metastasis by phosphorylating key regulators involved in cytoskeleton reorganization, such as Lim kinase (LIMK) [17,18] and the P41-ARC subunit of the ARP2/3 [19]. PAK1 activation also stimulates anti-apoptotic pathways, such as the Pak-Raf1-Bad [20,21] and NFkB [20] pathways, rendering PAK1 attractive as a cancer therapeutic target [22]. There has been a rapid expansion in the development of peptides as potential drugs for cancer therapy over the last decade [23].
HIV-1 TAT protein transduction domain-mediated delivery of macromolecules has emerged as an alternative approach for the internalization of proteins into the cell from the external environment [24]. PAK peptides have been also examined by two groups in different methods; 1) treatment of the PAK peptide (aa 11-23) that interacts with NCK [25]; 2) expression of PAK1 inhibitory domain (aa 83-149) [26].
The crystal structure of the Cdc42-PAK1 complex revealed that the CRIB domain of PAK interacts with Cdc42 by forming an intermolecular b-sheet between residues Y40-I46 of Cdc42 and I76-H83 of PAK but that this interaction seems to be disrupted by the presence of a b-bulge in PAK formed by the sequence 79 SDF 81 [4]. To get insights into the role of this sequence, here we investigated the effect of the mutation at S79, one of the three residues of the 79 SDF 81 sequence on the regulation of PAK1 activity. Our biochemical and cell biological studies have demonstrated that S79 plays a crucial role in the PAK1 interaction with Cdc42 and Rac1 and is required for PAK1-mediated malignant transformation of prostate epithelial cells. Thus, this study uncovers a previously unappreciated role of S79 in the regulation of PAK1 activity and demonstrates a novel concept for the activation of PAK1 by the GTPases.

Results and Discussion
PAK1 S79 Plays an Important Role in Autophosphorylation and Kinase Activities of PAK1 PAK1 interacts with Cdc42 and Rac via the CRIB domain (amino acid residues 74-88) [1]. A study indicated that 79 SDF 81 motif is positioned near the center of the CRIB domain and appears to disrupt the intermolecular b-sheet interaction between residues Y40-I46 of Cdc42 (blue) of Cdc42 and I76-H83 of PAK (red) [4] (Fig. 1A). Our sequence alignment showed that this motif is conserved only in higher eukaryotic organisms, suggesting diverse mechanisms for the regulation of PAK1 activity (Fig. 1B). Increased PAK1 activity is associated with autophosphorylation at specific sites, including S144, S199 and T423 [14]. To address whether S79 of PAK1 (PAK1 S79 ) is required for autophosphorylation and kinase activity of PAK1, we assessed phosphorylation states of these residues in PAK1 (WT) and PAK1 S79A by IP/ Western blot analysis and PAK1 kinase activity by in vitro kinase assay, respectively. Our results show that S79A mutation significantly decreases the phosphorylation of the three residues ( Fig. 1C) and kinase activity of PAK1 toward the PAK1 substrates MBP (myelin basic protein) and DLC1 (dynein light chain 1) peptide (Fig. 1D). PAK1 activation is stimulated by a variety of factors including epidermal growth factor (EGF) [16]. We found that S79A mutation markedly decreases EGF-induced PAK1 autophosphorylation at both S144 and T423 (Fig. 1E).

PAK1 S79 is Required for the Interaction of PAK1 with Rac1
Given that PAK1 activation is induced by the binding of the activated GTPase to the CRIB domain [4,5,6], we next examined S79A mutation effect on the PAK1 interaction with the Cdc42 and Rac1 GTPases. To this end, GFP-PAK1 and GFP-PAK1 S79A were coexpressed with Cdc42 or Rac1 in 293T cells, and their interaction was assessed by Co-IP/Western blot analysis. Wild type PAK1 was shown to interact with Cdc42 ( Fig. 2A) and Rac1 (Fig. 2B). However, the ability of PAK1 S79A to interact with the GTPases was markedly decreased; the binding affinity of PAK1 S79A for Cdc42 was reduced by ,3-fold ( Fig. 2A), whereas the Pak1 interaction with Rac1 was barely detectable (Fig. 2B). GST pull-down analysis also revealed a direct interaction between GFP-PAK1 (WT) and GST-Cdc42 (C) or GST-Rac1 (D) bound to GST-beads, whereas PAK1 S79A mutant has reduced affinity for both GTPases, for Rac1 in particular. However, we also found that S79D mutation does not affect in PAK1 activity towards MBP (Fig. 2E) and in the PAK1 interaction with Rac1 (Fig. 2F).  The S79A Mutation Impairs the Ability of PAK1 to Induce Changes in Cell Morphology and Motility PAK1 is translocated to the focal adhesions and membrane ruffles [27,28] and the sites of cortical actin remodeling [29] in stimulated cells. We examined the functional importance of PAK1 S79 by comparing the morphology and motility of PAK1 2/2 MEF (mouse embryonic fibroblast) cells expressing GFP-PAK1 and GFP-PAK1 S79A (Fig. 3A). Wild type MEF cells (PAK1 +/+ ) exhibited a bipolar fusiform shape (Fig. 3A, a-c), whereas PAK1 2/ 2 MEF cells displayed a more rounded morphology (Fig. 3A, d-f). Expression of GFP-PAK1 in PAK1 2/2 MEF cells restored the wild type cell shape (Fig. 3A, g-i), whereas GFP-PAK1 S79A expression did not rescue this defect (Fig. 3A, j-l). F-actin was colocalized with PAK1, as observed previously in Swiss 3T3 cells [28]; however, this colocalization was significantly reduced in MEF cells expressing PAK1 S79A (Fig. 3A and Fig. S1). MEF cells expressing GFP-PAK1 S79A exhibited 1.5,2 fold decrease in the ratio of length to width (L/W), compared with MEF cells expressing GFP-PAK1, whereas those cells expressing GFP-  (Fig. 3B). Wound healing migration assays showed that impaired ability of PAK1 2/2 MEF cells to migrate into, and close to, the wound was restored by expression of GFP-PAK1 but not of GFP-PAK1 S79A (Fig. 3C).
Next, we compared the ability of wild type and mutant (S79A) PAK1 proteins to confer a migration phenotype on the benign prostate RWPE-1 cells. To this end, RWPE-1 cells were infected with lentivirus expressing the vector plasmid, GFP-PAK1, or GFP-PAK1 S79A (S79A). Western blot analysis indicated no significant difference in the expression of GFP-PAK1 and GFP-PAK1 S79A in RWPE-1 cells (Fig. 4A a) ). Cell invasion assay showed that expression of GFP-PAK1, but not of GFP-PAK1 S79A , confers an invasive phenotype to RWPE-1 cells (Fig. 4A). We also examined cell morphology of RWPE-1 cells expressing PAK1 or PAK1 S79A . The formation of membrane ruffles was increased by ,2.5-fold (Fig. 4C) in RWPE-1 cells expressing GFP-PAK1 (Fig. 4B, d-f), compared with cells expressing GFP-PAK1 S79A (Fig. 4B, g-i) or control cells (Fig. 4B, a-c). These results are consistent with previous observations that PAK1 regulates the formation of membrane ruffles of Swiss 3T3 cells [28] and breast cancer cells [30]. We also found that RWPE-1 cells fill ,30% and ,60% of the wounded areas by expression of PAK1 and PAK1 S79A , respectively, at 24 h after scratching ( Fig. 4D and Fig. 4E). These observations demonstrate that S79 is crucial for PAK1-mediated cell migration.

A PAK1-inhibiting Peptide (TAT-PAK1 67-84 ) Blocks the Growth of the Prostate Cancer Cell Line PC3
Given that PAK1 activation is triggered by interaction with Cdc42 and Rac1, we examined whether PAK1 activity is reduced by a PAK1-inhibiting peptide. Towards this aim, a peptide containing the CRIB (PAK1 67-84 ) was chemically synthesized (Fig. 5A) and fused to the C-terminus of the TAT protein polybasic sequence to facilitate entry into cells as a protein transduction domain [31,32]. PC3 cells were treated with the peptide at low (2 mg/ml) and high (20 mg/ml) concentrations and were examined for cell growth by MTT assay. We found that treatment of the PAK1 peptide has ,2-fold inhibitory effect at high dose used (Fig. 5B). To confirm the significance of S79 in the activation process of PAK1 as described above, we also generated a TAT-PAK1 67-84 peptide with S79A mutation and tested its ability to inhibit PAK1 activation. Western blot analysis indicated that S144 phosphorylation is reduced by ,40% by the treatment of the PAK1 peptide but is not affected by the treatment of the PAK1 (S79A) peptide (Fig. 5C). We also examined the inhibitory effect of the PAK1-inhibiting peptide on the morphology PAK1 +/+ MEF cells (Fig. 5D). The bipolar fusiform shape of MEF cells (PAK1 +/+ ) was changed to a more rounded morphology by treatment of the PAK1-inhibiting peptide, whereas the treatment of the PAK1 (S79A) peptide has little effect on the morphology of MEF cells (Fig. S2).

Conclusion
The CRIB proteins such as PAK1-3 kinases [28], ACK tyrosine kinases [33,34] and the Wiscott-Aldrich-syndrom proteins (WASPs) [35,36] are activated by direct binding to Cdc42 [37]. While PAK binds to both Cdc42 and Rac, ACK and WASP do not bind to Rac [4]. Structural analyses indicate that the CRIB motif of all the three proteins make an intermolecularb-sheet interaction with the b2 strand of Cdc42 [4,38,39]. Thus, mutations of the amino acid residues that are evolutionarily conserved from human to Drosophila such as I75, S76 and P78 (for sequence alignment, see Fig. 1B) dramatically reduces the binding affinity of PAK for Cdc42 [40]. S79 is conserved only in higher eukaryotes and one of three residues ( 79 SDF 81 ) forming a bbulge that disrupts this interaction [4]. The structure of the Cdc42-PAK complex infers that S79 might interact with the V42 of Cdc42 [4] (Fig. 5E), whose mutation does not significantly affect the interaction of Cdc42 with PAK [39]. Hence, S79 is not essential for the PAK interaction with Cdc42. This may be in line with our finding that S79A mutation has little effect on the PAK1-Cdc42 interaction ( Fig. 2A-Fig. 2D). However, this mutation is shown to abolish the binding of PAK1 to Rac1, suggesting that PAK1 may bind to Cdc42 and Rac1 by different mechanisms and that S79 may play a key role in enabling PAK1 to distinguish Cdc42 and Rac1. This view is reinforced by the previous work that PAKs bind Rac1 with higher affinity than Cdc42. [41]. The crystal structure of the PAK1-Rac1 complex will help to elucidate the role of S79 in the interaction with and activation by Rac1.

Cell Culture
The RWPE-1 cells were grown in keratinocyte serum-free medium (K-SFM) containing bovine pituitary extract and epidermal growth factor, as described previously [42]. MEF, PC3, and 293T cells were cultured in RPMI 1640 or DMEM containing 10% FBS and penicillin/streptomycin at 37uC in a humidified atmosphere of 5% CO 2 . PAK1 wild type and PAK1 2/ 2 murine embryonic fibroblasts (MEFs) were kindly provided by Dr. Rakesh Kumar [43]. MEFs were isolated from day 13.5 wildtype or Pak1 2/2 embryos. Wild-type and Pak1 2/2 MEFs were immortalized with SV40 T antigen and were maintained in DME supplemented with 12% FBS. RWPE-1, PC3, and 293T cells cell lines used in this study were obtained from the American Type Culture Collection (ATCC; Rockville, MD).

Cell Invasion and Migration Assays
Cell invasion assay was performed using the cell invasion kit (Transwell Boyden's chamber with TranswellH Permeable Support Inserts Coated with CultrexH BME (basement membrane extract) Corning Costar) according to the manufacture's instruction. For the cell migration assay, confluent RWPE-1 or MEF cells were scratched with a P-200 pipette tip to cause wounding and subjected to the wound healing assay as described previously [42].

MTT Cell Proliferation Assays
For MTT cell proliferation assay, PC3 cells were cultured in 96well microplate. Cell growth was evaluated by replacing the culture media with 200 ml of 0.5 mg/ml MTT-media solution after incubation for 1-4 days. The absorbance was determined at 595 nm using a microplate reader (Bio-Rad Laboratories, iMark).