3.1. Introduction
p21-activated kinases (PAKs) have been associated with cell motility and morphology, yet it has been challenging to interrogate functions of individual PAK isoforms in living cells due to non-specific actions of PAK inhibitors and activation of compensatory pathways upon genetic manipulations. Here we engineer a genetically encoded, ligand-sensitive analogue of the isoform PAK1 that enables allosteric and selective kinase activation in living cells. We show the mechanism of allosteric control at a single-residue level through identified propagation pathways from the perturbation site to the active site. Our analysis on structural and dynamic features of the kinase domain also reveals alternate allosteric pathways that were exploited to generate versatile kinase switches. In order to capture consequences of PAK1 activation on cell motility, we turn on this switch in various living systems. Activation of PAK1 results in transient cell spreading in metastatic human breast cancer cells. Interestingly, in mouse hippocampal CA1 neurons, PAK1 activation leads to persistent dendritic spine enlargement. These results underscore the context-dependent roles of PAK1 in cell motility, and also suggest design routes to make various allosteric protein switches for controlling spatiotemporal dynamics of kinases.
p21-activated kinases (PAKs), consisting of six members (PAK1-6), are serine/threonine kinases involved in many cellular processes including cell growth, death, survival, proliferation, differentiation, motility and polarity (125). PAK is a key regulator of cytoskeleton networks, as its activity has been associated with protrusions (126-128), dissolution of stress fibers and loss of focal adhesions (129), control of adhesion turnover and strength (10, 129, 130). Such major cytoskeletal roles render PAK particularly relevant in various motility-driven biological processes including cancer metastasis (131), angiogenesis (132), synaptic transmission (24, 133, 134), and neurogenesis (135). However, these studies remain to be correlative; it is unclear how PAK dynamically contributes to cell motility, and the requirement of its kinase activity in motility remains controversial (125, 126, 136).
PAK inhibitors offer potential to interrogate dynamic roles of PAK in living systems, and to treat several disorders including certain types of cancer (131, 137) and schizophrenia (138). However, target specificity of these inhibitors is debated; thus, it remains uncertain whether a particular PAK isoform is responsible for a suggested mechanism. We decided to apply our RapR and uniRapR technologies in order to selectively activate a PAK1 isoform However, in contrast to the monomeric kinases we previously targeted (71, 139, 140), PAK is a homodimer in its inactive state (88), hence it is not obvious whether this method would be applicable to PAK. Here we engineer novel PAK switches and revealed the effects of its kinase activity on cell motility in breast cancer cells and hippocampal neurons.
3.2 Results and Discussion
Engineering and optimization of a dual chain PAK1 switch
We decided to focus on the PAK1 as a proof of concept, because it is the dominant isoform upregulated in human tumors, and it is one the three isoforms (PAK1-3) expressed in brain. Similar to other PAKs, PAK1 consists of a kinase domain and regulatory domains including p21-binding domain (PBD) for the localization and PAK inhibitory domain (PID) for the regulation of activity. In order to control directly the kinase activity, we targeted the kinase domain in our both dual and single chain PAK1 switch designs (Figure 3.1A and B). Dual-chain design has unique capabilities to turn on the switch at a particular subcellular location or to guide the switch to a target molecule. Single-chain design conveniences the delivery of the construct and guarantees equal expression of iFKBP and FRB. The insertion site for the kinases used in our previous studies indicates that our ligand sensitive allosteric domains (iFKBP and uniRapR) can be inserted into the loop between residues 288 and 293 in PAK1. This loop is not evolutionary conserved, indicating it is not essential for the function of protein (Figure 3.2). Also, this loop neither interacts with the regulatory domain nor with the dimerization interface (Figure 3.1C). Our structural analysis also showed that this loop is surface exposed and it does not interact with any other structural elements in the protein (Figure 3.3A), indicating a proper insertion site for our allosteric domains.
(A) Dual chain (rapamycin regulatable or RapR) PAK1 switch was built by inserting insertable FKBP (iFKBP) domain into the catalytic domain. The system requires co-expression of FRB. (B) Single chain (unimolecular rapamycin regulatable or uniRapR) PAK1 switch was built by inserting insertable uniRapR domain into the catalytic domain. This system does not require co-expression of FRB. (C) Crystal structure of PAK1 in inactive state. Auto-inhibitory domain (AI) and p21 binding domain (PBD) are shown with salmon and green, respectively. Insertion sites are red residues shown with black arrows. (D) Rapamycin dependent activity of constitutively active (CA) PAK1, kinase-dead (KD) PAK1, RapR-PAK1, and uniRapR-PAK1. (E) Area and (F) perimeter of MDA-MB-231 cells that stably express uniRapR-PAK1 before and after addition of rapamycin.
We first sought to design a dual chain PAK1 switch, and replaced the insertion loop with the iFKBP domain (Figure 3.1A and Figure 3.3). In order to increase the activation effect, we introduced phospho-mimetic S422D/T423E mutations into PAK1 to render it constitutively active. We substituted residue 288 in the insertion loop with the iFKBP and a double GPG linker, which was used for other RapR kinases. We co-expressed this construct (288 GPG) with FRB in HEK293T cells. Following immunoprecipitation of PAK1, we tested the kinase activity using a PAK1 substrate, MEK1, which is phosphorylated at residue S298 by PAK1 Figure 3.1. Engineering genetically encoded PAK1 switches
(A) Residues used for insertion are not conserved. (B) Conservation of the kinase domain, represented by sequence entropy, mapped on the structure.
Next, we targeted the next residue 289 in the same loop and substituted this residue with iFKBP either with a G-linker or GPG-linker. Remarkably, we observed almost full PAK1 activation upon addition of rapamycin (Figure 3.3B). FRB was present in pull-down samples, indicating the binding of FRB to PAK1-iFKBP. We further increased the dynamic range of activation by substituting entire loop with iFKBP. We termed this version as rapamycin regulatable PAK1 (RapR-PAK1). As a control experiment, constitutively active (CA) PAK1 alone did not respond to rapamycin, whereas kinase-dead (KD) PAK1 alone was inactive in the absence or presence of rapamycin (Figure 3.3B). All these experiments demonstrated that Figure 3.2. Sequence conservation of PAK1 kinase domain
insertion of iFKBP domain into the loop between residues 288 and 293 generates a robust PAK1 switch.
(A) Structure of PAK1 dimer and residues in the insertion loop. (B) Biochemical in vitro test of various RapR-PAK constructs.
Single chain PAK1 switch in triple negative breast cancer cells
We made a single chain version of RapR-PAK1 by replacing iFKBP with the uniRapR domain (uniRapR-PAK1). We directly compared RapR-PAK1 and uniRapR-PAK1 by monitoring the phosphorylation state of endogenous MEK1 upon addition of rapamycin into the cell media. Similar to the behavior of RapR-PAK1, uniRapR-PAK1 was inactive without rapamycin and rapidly activated by rapamycin (Figure 3.1D). We further increased the Figure 3.3. Insertion loop and optimization of linkers
dynamic range of uniRapR-PAK1 by introducing another mutation L107F (141) that blocks dimerization and enhance activity of PAK1. These results showed that both dual and single chain PAK1 switches could be rapidly activated in living cells by rapamycin.
PAK1 was identified as a breast cancer oncogene that transforms breast epithelial cells (142). Moreover, overexpression of mutant PAK1 was shown to increase migration rates of breast cancer cells (143). In order to demonstrate direct effects of PAK1 kinase activity in breast cancer cells, we expressed uniRapR-PAK1 in highly metastatic triple-negative breast cancer cells MDA-MB-231. We measured the area and perimeter of cells after 5, 20, and 60 minutes of rapamycin addition. Remarkably, cell area and perimeter had a transient increase upon addition of rapamycin (Figure 3.1E and F). After such a rapid spreading, cells went back to their original morphology at the 20-minute time point. Control cells exhibited no response to rapamycin. These results indicated that catalytic activation of PAK1 resulted in transient spreading; suggesting metastasis signaling circuits have additional requirements for persistent spreading in addition to PAK1 activity.
PAK1 activation results in persistent dendritic spine enlargement
Surprising transient effects of PAK1 kinase activation in triple negative breast cancer cells led us to investigate PAK1 kinase activity in other living systems. PAK1 is expressed in axons and dendrites in hippocampal and cortical neurons (144, 145), and constitutively active mutant is enriched in dendritic spines (146). Our previous findings in mice hippocampus showed that prolonged photo-activation of upstream Rac1, which is an activator of PAK1, leads to shrinkage of dendritic spines (147). Thus, we asked whether persistent activation of PAK1 leads to same morphological change.
(A) Image of RapR-PAK1-Venus in a dendrite. (B) A series of RapR-PAK1-Venus images in a dendrite before and after stimulation with rapamycin (2.5 µM), which was applied in the period between 0 and 15 min. (C) Volume changes of two spines (#1 and #2) indicated in (B). The changes were estimated from the total fluorescence of RapR-PAK1-Venus in the spines. (D) Mean time courses of spine volume obtained from 33 or 13 spines in either RapR-PAK1- Venus or EGFP along with FRB-mCherry transfected neurons. Error bars indicate SEM. (E) Statistical comparisons of the effects of rapamycin (2.5 µM) shown in (D). *p < 0.05, **p < 0.01.
We expressed RapR-PAK1-EGFP in hippocampal CA1 neurons (Figure 3.4A), and surprisingly observed that activation of PAK1 led to persistent dendritic spine enlargement (Figure 3.4B). Although each spine responded to rapamycin with different kinetics (Figure Figure 3.4. Enlargement of dendritic spines induced by rapamycin
3.4C), there was an overall persistent increase in the spine volume (area of change 60% ± 15%, n = 35 spines), whereas neuron expressing only EGFP did not respond to rapamycin (n = 13 spines) (Figure 3.4D and E). Based on these results, we concluded that activation of PAK1 leads to persistent dendritic spine enlargement.
Allosteric propagation in PAK1 can be maintained through alternative pathways What are the rules to make a kinase switch? We hypothesize that loops that move in concert with the active site can be an insertion site for the allosteric domain, as perturbation of the insertion site would be propagated to the active site. Additionally, we select loops that are surface exposed or not in contact with the rest of the protein. We analyzed the molecular dynamics of PAK1 kinase using discreet molecular dynamics (111, 123). Indeed, motions of the insertion loop (L1) correlate with the catalytic site, particularly the ATP binding site (Figure 3.5A). Using the same analysis, we also identified another loop (L2) whose motions correlate with the motion of ATP binding site. Similar to L1, this loop is also solvent exposed and is not evolutionarily conserved (Figure 3.5B).
In order to test whether L2 loop is permissive to generate an allosteric switch, we substituted residues G337 and D338 in L2 with uniRapR domain. Similar to the original PAK1 switch (uniRapR-PAK1-L1), the uniRapR-PAK1-L2 switch responded to rapamycin immediately (Figure 3.5C). We further reduced the slight off-state activity by removing two more residues from the loop, V336 and E339 (Figure 3.5C). These results along with the results of linker optimization of the first switch showed that the activity range can be adjusted through the linkers. Off-state activity can be reduced by shortening the loop or removing residues from the insertion loop, and off-state can be increased by elongating the loop using flexible residues.
Overall, a PAK1 switch can be generated by inserting uniRapR into the loops whose motions correlate with the motions of catalytic site.
(A) Correlative motions of residues in PAK1 kinase domain. Correlation map of the kinase domain. Red denotes correlation, while blue denotes anti-correlation between two residues. b. Network representation of kinase domain based on contacts (<5.5Å). Gray nodes represent residues; edges represent the contacts. L1 is the canonical insertion site and L2 is newly identified insertion site. Red arrows represent the propogation pathways to ATP binding site (yellow node). (C) Insertion of uniRapR to both insertions sites confer rapamycin-mediated control. In the second version of L2 based switch (uniRapR-PAK1-L2-v2), loop residues from PAK1 removed, resulting in a tighter control. (D) Suggested mechanism of L1 based switch. (E) Suggested mechanism for L2 based switch. (F) Summary of the mechanisms of PAK1 switches.
How is the propagation transmitted from the insertion loop to the active site? We hypothesize that residues that move in concert in the kinase domain may constitute the same Figure 3.5. Computational design to generate versatile PAK1 switches
allosteric path, which can be responsible for the propagation. In order to track potential propagation pathways, we performed a network analysis by converting motion dynamics of residues into an undirected network, in which nodes represent residues, edges represent contacts, and edge weights are assigned based on correlation coefficients calculated by molecular dynamics simulations (Figure 3.5B).
Co-evolved residues of PAK1 kinase domain were identified using MISTIC (148).
The shortest path from insertion loops L1 or L2 to the catalytic site was calculated using Dijkstra’s algorithm (149). We observed that the propagation from L1 is transmitted through the strands located between residues G293-Q300 and G282-D289 (Figure 3.5C). Interestingly, such intra-strand propagation is not present in the case of L2, where the perturbation is propagated in an inter-strand manner between strands E91-E97, G45-N54, G34- Figure 3.6. Residues that are co-evolved with L1 and L2 loops
D41, and E26-D41 (Figure 3.5D and E). As an alternative approach, we analyzed potential allosteric pathways using co-evolution of residues (148). Identification of residues that co- evolve with the catalytic site indicated a similar inter-strand pathway (Figure 3.6). All these results showed that perturbation is propagated from our canonical (L1 insertion), which was used in our previous work, and non-canonical (L2 insertion) insertion loop via intra-strand and inter-strand manner, respectively.
3.3 Conclusion
Here we engineer PAK switches that are turned on by rapamycin or its non- immunosuppressive analogs. We previously showed that non-immunosuppresive analogs also bind both uniRapR and iFKBP domains, thus we also expect that our PAK1 switches can be activated by such analogues. We classify our PAK1 switches as “conformational switches”, meaning that an inducer controls the conformation of the protein. This type of tool has unique advantages; such as substantially low activity before being switched on. In contrast to many other tools, that are designed to control localization of protein activity rather than its conformation, can be used for certain proteins which are only active in certain subcellular region. However, PAK1 is involved in many subcellular regions in the cell, and our approach does not intervene with its normal localization. An alternative way to study PAK1 in rapid and dynamic processes would be using PAK inhibitors. Like other kinase inhibitors, many PAK inhibitors, which are ATP-competitive molecules, are not very selective. An allosteric inhibitor, IPA-3 was designed to bind amino-terminal regulatory domain group 1 PAKs, yet undesired toxic effects in cells and unselective binding with PAK1-3 renders these inhibitors problematic.
We focus on PAK1 as a target protein, because its role in cytoskeleton remains controversial, and to our knowledge, there is currently no PAK1 switch in the literature. Many of the previous studies included overexpressed constitutively active or dominant negative PAKs. These studies suggest that overexpression of constitutively active PAK leads to large and polarized lamellipodia, whereas expression of dominant negative PAK leads to multiple random lamellipodia in fibroblasts (127). Nevertheless, overexpression of constitutively active kinases can increase intracellular signaling above the physiological basal state. On the other hand, studies that used acute activations through injection of constitutively active PAK1 into the cells report rather different phenotypes (126, 136). Because PAK1 is upregulated in metastatic breast cancer, we first tested phenotypic effects of PAK1 in highly metastatic MDA- MB-231 cells. PAK1 activation caused transient spreading, suggesting that other pathways are required for a persistent metastatic behavior of these cells.
In addition to its association to metastatic breast cancer, PAK1 is involved in basic neuronal behaviors. PAK1 is implicated in synaptic transmission, spine morphology, and several pathological conditions such as mental retardation and schizophrenia. but consequences of its kinase activity is unclear. The kinase activity of PAK is important, because PAK inhibitors have been shown to ameliorate schizophrenia-associated dendritic spine deterioration (150). However, these inhibitors are not isoform specific, thus it remains unclear which isoform is responsible for the observed effect. Upstream of PAK1, GTPase Rac1 has been suggested as a key regulator in modulation of neuronal activity. We and other previously showed that activation of Rac1 in vivo leads to spine shrinkage in hippocampus (147, 151). Here we find that activation of PAK1 leads to spine enlargement in CA1 hippocampal pyramidal neurons. This observation suggests that Rac1 mediated memory erasure is mediated
by PAK1 independent mechanisms. These results may give insight into the roles of PAK1 in long-term-potentiation, learning and memory.
Our analysis on propagation mechanisms revealed that the perturbation is allosterically propagated through the beta strands located between G293-Q300 and G282-D289 in our canonical switch designs. This observation is expected as destabilization of a loop in a structural unit such as beta-strand perturbs other residues in the same strand. However, we observed an inter-strand allosteric propagation in our second type of design. Such an allosteric propagation is meaningful, because correlated motions in B-sheets have been suggested to be fundamental for allosteric regulation of protein function (152). Understanding propagation mechanisms in our allosteric switches will be helpful to generate novel switches not only for different kinases but also for other protein families.
3.4 Materials and Methods DNA constructs
All restriction enzymes were purchased from New England Biolabs. Point mutations and insertions of iFKBP and uniRapR sequences into PAK1 cDNA were done with Quikchange mutagenesis kit (Stratagene). Retroviral plasmids were generated by cloning uniRapR-PAK1-mCherry into pBABE-Tet-Off-puro plasmid (Clontech).
Biochemical characterizations
Anti-myc antibody were purchased from Millipore. Anti-pMEK-S298 and anti- MEK1/2 from X. Anti-GFP (JL8) was purchased from Clontech. Rapamycin was purchased from Sigma. HEK293 cells were transfected with 1 µg of DNA constructs using Fugene6
reagent (Roche). After 24 hours of transfection, cells were treated with either rapamycin or an equivalent volume of ethanol for 30 minutes. Cells were lysed with lysis buffer (20 mM HEPES-KOH, pH 7.8, 50 mM KCl, 100 mM NaCl, 1 mM EGTA, 1% NP40, 1mM NaF, 0.1 mM Na3VO4, and Roche protease inhibitor cocktail). Endogenous phosphorylation of MEK
was detected by western blotting. For immuniprecipitation experiments, cleared lysates were incubated with protein-G beads attached to anti-GFP antibody for 2 hours. The beads were washed three times with wash buffer (20 mM HEPES-KOH, pH 7.8, 50 mM KCl, 100 mM NaCl, 1 mM EGTA, 1% NP40), and then with kinase buffer (25 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, 0.5 mM EGTA, 0.005% BRIJ-35) for phosphorylation assays. Purified ATF2 and
ATP (0.5 mM) in 40 µL of kinase buffer were mixed with bead suspension for the reaction for 10 minutes. Phosphorylated MEK was detected in pull-down samples by western blotting.
Imaging of MDA-MB-231 cells
After allowing 2 days without doxcyline in the media, cells were seeded at 10,000 cells per well in DMEM with puromycin on fibronectin coated coverslips (10µg/ml). The next morning (about 6 hours before the experiment), cell media was replaced with to the DMEM (2% FBS) with 450µl in each well of a 4 well plate. Cells were incubated with 500 nM of rapamycin for 5, 20, and 60 minutes. Cells were then fixed in 4% PFA (in PBS) and stained