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Integrin-linked kinase (ILK) plays a pivotal role in connecting transmembrane receptor integrin to the actin cytoskeleton and thereby regulating diverse cell adhesion-dependent processes. The kinase domain (KD) of ILK is indispensable for its function, but the underlying molecular basis remains enigmatic. Here we present the crystal structure of the ILK KD bound to its cytoskeletal regulator, the C-terminal calponin homology domain of α-parvin. While maintaining a canonical kinase fold, the ILK KD displays a striking pseudo-active site conformation. We show that rather than performing the kinase function, this conformation specifically recognizes α-parvin for promoting effective assembly of ILK into focal adhesions. The α-parvin-bound ILK KD can simultaneously engage integrin β cytoplasmic tails. These results thus define ILK as a distinct pseudokinase that mechanically couples integrin and α-parvin for mediating cell adhesion. They also highlight functional diversity of the kinase fold and its “active” site in mediating many biological processes.
The communication between transmembrane integrin receptors and the actin cytoskeleton is fundamental for cell migration, spreading and differentiation. Integrin-linked kinase (ILK) is one of the few essential and evolutionarily conserved mediators in this communication process (Wu, 2004; Legate et al., 2006). Originally identified as an integrin β cytoplasmic tail (CT) binding protein, ILK was thought to be composed of an N-terminal ankyrin repeat domain, a middle pleckstrin homology (PH)-like motif, and a C-terminal kinase domain (KD) (Hannigan et al., 1996). Cell biological and biochemical data have continued to mount, establishing ILK as a key molecule in coupling integrins with the actin cytoskeleton (for review, see Legate et al., 2006). The importance of such ILK-dependent coupling has been underscored by several elegant genetic analyses in Drosophila (Zarvas et al., 2001), Caenorhabditis elegans (Mackinnon et al., 2002), and mice (Sakai et al., 2003), which showed that depletion or dysregulation of ILK leads to severe defects in the integrin-containing cytoskeleton structure and cell adhesion dynamics. Despite the overwhelming information about the biological importance of ILK, the precise molecular underpinning of ILK function remains elusive. In particular, while ILK has been widely claimed to act as a signaling serine-threonine kinase to trigger diverse integrin signaling pathways (Hannigan et al., 2005), its catalytic function has been under significant debate (Hannigan et al., 2005; Legate et al., 2006) since genetic analyses indicated that ILK kinase activity may not be required for normal tissue development and function (Zarvas et al., 2001; Mackinnon et al., 2002; Sakai et al., 2003; Dai et al., 2006; Kanasaki et al., 2008). Examination of the ILK KD primary sequence suggested a pseudokinase function of the protein with some variations in the putative catalytic site (Boudeau et al., 2006; Scheeff et al., 2009), but many cell-based analyses reported that ILK was capable of directly phosphorylating diverse substrates including a generic substrate myelin basic protein (MBP) and physiological targets (for review, see Hannigan et al., 2005) such as integrin β1 CT (Hannigan et al., 1996), myosin light chain kinase (Deng et al., 2001), β-parvin (Yamaji et al., 2001), and Akt/PKB (Persad et al., 2001). The ILK kinase activity was also shown to be significantly enhanced by several co-factors including PIP3 (Delcommenne et al., 1998), the C-terminal calponin homology domain (CH2) of α-parvin (Attwell et al., 2003), and the actin monomer sequestering protein, thymosin β4 (Bock-Marquette et al., 2004; Fan et al., 2009).
Although variations in the primary sequences of the catalytic motifs are often used to predict whether a protein has catalytic function or not, recent 3D structural analyses indicated that this sequence-based approach is not always valid. For example, WNK kinase, which lacks the conserved catalytic lysine residue in the subdomain II (replaced by C250) that corresponds to K72 in protein kinase PKA), was found to use K233 in its β2 strand as an alternative catalytic lysine (Min et al., 2004). Also, CASK, which was initially thought to be catalytically inactive due to the lack of the DFG aspartate that can coordinate a functional magnesium ion (Mg), was shown to be catalytically active in an Mg-independent manner (Mukherjee et al., 2008). Given the highly conflicting data yet central importance of ILK in biology, a definitive structure-function analysis on ILK KD is necessary to define the precise mechanism of ILK function. To this end, we have determined the crystal structure of the ILK KD bound to its putative activator α-parvin CH2. Our structure revealed a distinct pseudo-active site in ILK thus defining it as a pseudokinase. More detailed analysis demonstrated that ILK lacks intrinsic kinase activity, yet utilizes its pseudo-active site to recognize α-parvin for focal adhesion targeting. While pseudokinases are emerging as an important class of protein regulators (Boudeau et al., 2006), exactly how they function remains largely unknown. Our results provide significant insight into this class of proteins. In particular, the pseudo-active site-mediated target binding not only helps to elucidate the mechanism of ILK but also sheds light upon the functional diversity of the kinase fold and its “active” site in mediating diverse cellular events.
A series of constructs comprising the C-terminal KD of ILK was examined for expression but produced highly insoluble ILK aggregates, precluding further structural analysis. Based on the previous reports that α-parvin CH2 (therein CH2) binds to ILK KD (Tu et al., 2001) and activates it (Attwell et al., 2003), we constructed a bicistronic coexpression system (Tan, 2001) to co-express CH2 with a human ILK fragment (183-452) containing the putative PH domain and KD (Hannigan et al., 1996). This approach yielded a soluble and mono-dispersed complex suitable for the structural analysis. We crystallized and solved the structure of the complex at 1.8 Å resolution. We also co-crystallized the complex in the presence of ATP and Mg and solved the structure at 2.0 Å resolution (Table 1). The structures are well defined except for the short N-terminal region in CH2, which exhibits poor density maps with relatively higher temperature factors (B-factors) compared to the overall values. This region is known to bind to another partner paxillin LD1 motif (Wang et al., 2008; Lorenz et al., 2008).
The overall fold of ILK KD is very similar to those of known kinases and contains characteristic bilobal domains (Knighton et al., 1991), namely, the N-terminal lobe that folds into a five-stranded anti-parallel β-sheet flanked by a short and a long (αC) α-helix, and the C-terminal lobe that contains a bundle of α-helices and a short pair of anti-parallel β-strands (β7-β8) (Figs. 1A and S1A). ATP is situated in a nucleotide-binding cleft between the two lobes (Figs. 1A and S1A) similar to the ATP binding site in other kinases (Manning et al., 2002) (Figs. S1B and S1C). Superposition of the ILK KD structure (ATP-bound form) onto the nucleotide-bound active conformation of PKA, a representative serine-threonine kinase (PDB entry 1ATP) reveals a similar global architecture with an RMSD of 2.4 Å (21% identity) (Fig. S1D). The well-defined ATP molecule in the ILK structure was surprising since previous sequence-based analysis suggested that ILK may not bind ATP due to a dramatically altered ATP binding loop (P-loop) where the well-conserved glycine-rich GXGXXG motif (X denotes any residue) in Ser-Thr kinases is replaced by a non-glycine-rich NENHSG motif in ILK (Scheeff et al., 2009). It was equally surprising that ATP was not hydrolyzed in the ILK structure since even inactive kinases such as MEK1 hydrolyze ATP into ADP (Fischmann et al., 2009) - the first step in the kinase reaction. However, despite the similar binding site, ATP in ILK has an unusual binding mode as compared to known kinases such as PKA: (i) ATP coordinates only one Mg in ILK whereas two Mgs are bound to ATP in PKA to facilitate catalysis (Fig. 1B). (ii) The tri-phosphate orientations of ATP, especially the β-γ phosphate groups in ILK are drastically different from those in PKA (Fig. 1C). Notably, the side chain of K341 from the activation loop of ILK (corresponding to glycine in the well-known DFG motif, see Fig. S1E) protrudes against the ATP phosphate groups and coordinates the γ-phosphate of ATP (Fig. 1B). Such distinct coordination might facilitate the distinct γ phosphate orientation as compared to that in PKA (Fig. 1B). Also, K220 in ILK bridges α- and γ-phosphates of ATP, whereas the equivalent lysine residue in known kinases (K72 in PKA) coordinates α and β-phosphates of ATP (Figs. 1D and S1F). (iii) ATP bound to the P-loop of ILK, is far away from the putative catalytic loop (Figs. 1D and 1E) whereas in known kinases, ATP, especially the γ-phosphate, is very close to the catalytic loop for catalysis (Figs. 1D and 1E). The Gly-rich P-loop is conformationally flexible to allow the bound ATP to interact with the catalytic loop (e.g., PKA in Fig. 1E). By contrast, the non-glycine-rich P-loop in ILK appears to lack such flexibility as indicated by low temperature factors, and thus no movement was observed with and without ATP (Fig. 1E). This lack of flexibility explains why the bound ATP in ILK remains distant from the catalytic loop (Fig. 1E).
The structure-based sequence alignment of ILK with representative kinases reveals that the catalytic loop in ILK diverges severely with significant insertion/deletion and absence of multiple catalytically important residues (Fig. S1E). In particular, our structure shows that the invariant catalytic base aspartate (D166 in PKA) in the hallmark HRD motif is replaced by a neutral residue A319 in ILK KD (Fig. 1E). This aspartate in PKA is known to properly orient the γ-phosphate and to accept a substrate hydroxyl moiety for phospho-transfer (Madhusudan et al., 1994; Zheng et al., 1993) (Fig. 1B). However, the corresponding residue A319 in ILK KD is not only hydrophobic but far away (~9-10Å) from the γ-phosphate of ATP (largely due to the inflexibility of the ATP-bound P-loop, see Fig. 1E) as compared to PKA (Figs. 1B and 1E). Inspection of the structure revealed no structurally alternative Asp or any similar and conserved residues near the catalytic loop (Fig. 1D). The significance of the conserved Asp is underscored by a recent study of the pseudokinase STRADα, in which the corresponding Asp is replaced by a serine (Baas et al., 2003). Other notable changes of the catalytic loop in ILK include N321, which corresponds to the catalytic lysine K168 in PKA that is involved in phospho-transfer, and S324, which corresponds to the catalytic aspargine N171 in PKA that coordinates the second Mg (Fig. 1E) and forms hydrogen-bond with the backbone carbonyl of D166. Here, although S324 hydroxyl group makes similar hydrogen bond with the backbone carbonyl of corresponding A319 in ILK, it does not coordinate with Mg since the second Mg is absent. Together with the above finding that the ATP γ-phosphate is geometrically far away from the catalytic loop (Fig. 1E), the deviations in the catalytic core indicate that ILK cannot perform the catalysis as a conventional kinase. The unusual structural arrangement also provides a strong clue as to why ATP was not hydrolyzed by ILK.
The activation loop of protein kinase is known to dynamically regulate kinase activity, especially substrate entry into the catalytic core. The activation loop of ILK spans from the DVK motif (residue 339-341 that corresponds to the DFG motif in known kinases) to the APE motif (residues 357-359 that corresponds to the C-terminal end of the P+1 loop) (Nolen et al., 2004) (Fig. S1E). Strikingly, the activation loop in ILK is significantly short and also lacks a highly conserved phosphorylation site known to regulate the conformation of the activation loop in conventional kinases (e.g., T197 in PKA) (Figs. 2A and S1E). The activation loops in typical kinases are disordered and become ordered upon phosphorylation, thus transitioning a kinase from an inactive to an active state (Nolen et al., 2004). Conversely, the activation loop in the ILK structure is well ordered without any phosphorylation (Figs. 1A and and2A).2A). Notably, the mean B-values of the ILK activation loop are relatively low at 19.3 Å2 compared to 21.7 Å2 for the whole ILK KD, indicating that this segment is quite rigid. This is likely due to the extensive hydrophobic and hydrophilic interactions between the activation loop and the N-lobe containing the αC helix (Fig. 2B). In particular, the side chain hydroxyl oxygen of S343 makes a hydrogen bond with the side chain of E238 in the αC-helix, and the two phenylalanine residues (F342 and F344) in the activation loop that sandwich S343 pack tightly against a cluster of hydrophobic residues, L222, V224, W227, and L267 in the N-lobe core and F235 and L242 in the αC-helix (Fig. 2B). Note that S343 was proposed to be a phosphorylation site for ILK activation (Persad et al., 2001) but our structure shows that S343 hydroxyl group is buried in the activation loop/N-lobe interface (Fig. 2B) and important for the structural integrity. Thus, the side chain of S343 is not freely accessible for phosphorylation contrasting to that observed in T197 in PKA for inducing the kinase activation. No direct experimental evidence has been reported for the S343 phosphorylation. Furthermore, a phosphorylation-mimic S343D mutation caused no effect on the ILK activation (see below).
The substitution of the canonical DFG in functional kinases with DVK in ILK also makes the activation loop distinct. The side chain conformation of D339 in the DVK motif of ILK is markedly different from those in the DFG motifs of typical kinases such that D339 only coordinates to one metal whereas the aspartate in DFG coordinates two metal ions (Fig. 1B) to promote the catalysis. Two residues are inserted immediately preceding D339 (Fig. S1E), which may lead to the distinct side chain orientation of D339 and unusual metal coordination involving this residue. More strikingly, ILK lacks the DFG glycine that allows the DFG to flip in (active) or out (inactive) during kinase activation (Fig. S2). As a result, the DVK motif of ILK appears to be locked in a “flipped in” position with or without ATP (Fig. S2), a structural feature consistent with the rigidity of ILK activation loop.
Structures of several atypical Ser/Thr kinases have been reported including Titin (Mayans et al., 1998), WNK (Min et al., 2004), and CASK (Mukherjee et al., 2008). Titin and WNK have remarkable ways to compensate for missing catalytic residues by alternative residues/motifs, i.e., the EFG glutamate in Titin acts as the DFG aspartate in typical kinases (Mayans et al., 1998) and the K233 residue in the β2 strand of WNK acts as the catalytic lysine equivalent to K72 in PKA (Min et al., 2004). CASK lacks two conserved Mg binding residues (corresponding to N171 and D184 in PKA) (Fig. S1E) and thus it functions as an Mg-independent kinase that selectively phosphorylates neurotoxin (Mukherjee et al., 2008). Importantly, these three kinases are atyptical because of an altered Mg binding site, but all retain the glycine-rich P-loop, the well-conserved catalytic loop, and very similar activation segment (Fig. S1E), which seem to be sufficient for kinase activity. By contrast, these hallmark features are significantly disrupted in ILK, i.e., the unusual ATP binding configuration, severely impaired catalytic core, and a short and rigid activation loop. No spatially conserved residues/motifs in the ILK structure were found to possibly compensate for these non-permissive catalytic features (Fig. 1D).
Although ILK has been widely regarded as a key serine-threonine kinase in integrin signaling (for reviews, see Hannigan et al., 2005; Legate et al., 2006), the above structural findings strongly suggested that ILK may be a pseudokinase, which prompted us to critically re-evaluate the ILK catalytic activity. The previously claimed ILK kinase activity was largely based on the cell-based assays or partially purified ILK. We thus decided to first perform kinase assays using purified recombinant ILK. As mentioned above, ILK alone is not soluble, so we co-expressed full-length ILK with PINCH LIM1-2 that binds to the N-terminal ankyrin repeat domain of ILK but does not affect the target binding and putative catalytic activity of the ILK KD (Chiswell et al., 2008; Yang et al., 2009). Using the PINCH-LIM1-2-ILK complex purified from bacteria, we observed no kinase activity on previously reported substrates including myelin basic protein (MBP, generic kinase substrate) (Hannigan et al., 1996; Kim et al., 2009), integrin β CT (Hannigan et al., 1996), and CH2 (Yamaji et al., 2001) (Fig. 3A). Recombinant ILK from commercial source (Randox Life Sciences, Inc) had no kinase activity either (data not shown). In comparison, bacteria-purified PKA and MEK kinases phosphorylated MBP very potently (Fig. 3A). We next asked if ILK can be activated via mutation or co-factors. Previous cell-based studies showed that ILK may be activated by phosphorylation-mimic S343D mutation (Persad et al., 2001) or co-factors including PIP3 (Delcommenne et al., 1998) and α-parvin CH2 (Attwell et al., 2003). However, purified ILK exhibited no activity upon S343D mutation or other potentially activating mutations or addition of the above co-factors (Fig. 3B). In fact, PIP3 did not even bind to ILK (data not shown) and the previously suggested PH motif is an integral part of the P-loop in our structure (Figs. S3A-S3C). Varying Mg concentrations in the kinase assays also had no effect. We next sought to perform kinase assays using endogenous ILK purified directly from chick-tissue (Deng et al., 2001). Although the chick tissue-purified proteins containing ILK phosphorylated generic MBP as previously demonstrated (Deng et al., 2001) (Fig. 3C), it had no effect on previously suggested physiological substrates of ILK including integrin β CT (data not shown) and Akt (Fig. 4). Furthermore, the MBP phosphorylation was not enhanced at all by CH2 that was reported to be an ILK activator (Attwell et al., 2003) (Fig. 3C). In fact, as we show below, CH2 binds tightly to the putative substrate site (the P+1 loop and G-helix), and thus its addition to the kinase reaction would inhibit the phosphorylation of MBP if the effect were directly mediated by ILK. However, no inhibitory effect was observed (Fig. 3C).
The most widely studied ILK target is Akt (for review, see Hannigan et al., 2005), and ILK-mediated Akt activation was shown to be enhanced by the actin-monomer sequestering protein thymosin β4 (Bock-Marquette et al., 2004; Fan et al., 2009). We thus performed kinase assays to specifically examine this effect. As shown in Fig. 4A, purified recombinant ILK and ILK S343D mutant showed no effect on Akt phosphorylation in the absence and presence of thymosin β4. Endogenous ILK purified from chick tissue also showed no effect on Akt in the absence and presence of thymosin β4 (Fig. 4B). In fact, in pull-down assays, we failed to see any direct interaction between thymosin β4 and ILK (data not shown), suggesting that the thymosin β4 effect on Akt (Bock-Marquette et al., 2004; Fan et al., 2009) might be indirect. Finally, we attempted to repeat the previously reported effect of ILK on Akt using co-immunoprecitated ILK (Persad et al., 2001). However, ILK co-precipitated from HEK 293 cells expressing FLAG-full-length ILK also had no effect on the Akt phosphorylation (Figs. 4C and 4D). It remains to be determined what factors caused the discrepancy between our assay and previous assays (Persad et al., 2001). It is possible that co-precipitation assays might involve an unknown kinase associated with ILK. Consistently, a large >500 kDa supramolecular complex containing ILK was reported to phosphorylate Akt, but some unknown components in this complex, not the co-precipitated ILK, was found to be responsible for the effect (Hill et al., 2002). Interestingly, we found that ILK is always co-purified with α-parvin and PINCH as a tight ternary complex from the tissue extracts (Fig. S4), supporting an emerging theme that ILK forms a functional heterotrimer in cells to mediate cytoskeleton assembly and cell adhesion (Wu, 2004; Legate et al., 2006).
Given the above structural and functional data that indicated ILK KD is catalytically inactive and may act as an adaptor, a key question is then how exactly ILK KD exerts its adapting function via its kinase fold. It was also puzzling to us why α-parvin or α-parvin CH2 had no effect on ILK activity (Figs. 3 and and4)4) given the previously identified role of CH2 as an ILK activator (Attwell et al., 2003). We thus examined the structure of the ILK KD-CH2 complex in detail, which revealed strikingly that CH2 recognizes a protruding surface in the C-lobe of the ILK KD that comprises a pair of α-helices (αEF-helix; αG-helix) and a small part of the C-terminal activation loop (P+1) in the ILK KD (Fig. 5A). The interface is quite large with the buried surface area of ~1,900 A2 that is a typical range for the high affinity complex (Lo Conte et al., 1999) (Figs. 5B and S5A-S5C). The corresponding αG-helix and the C-terminal activation loop in ILK are parts of the active site in known kinases, which are involved in substrate or regulator binding (Song et al., 2001; Kim et al., 2005; Dar et al., 2005). Such binding mode is similar to conventional kinase-target interaction as exemplified by PKR-eIF2α (kinase-substrate) and PKA-RIα (kinase-regulator) (Figs. S5D-S5G). However, the ILK-α-parvin interaction is highly specific since the residues in ILK that contact CH2 are not conserved in other kinases (Fig. S1E). In particular, the M402-K403 residues in the ILK G-helix that make the most extensive contact with CH2 (Fig. 5C) are not conserved in other kinases. To further elucidate the importance of this specific interaction, we designed a double mutation (M402A/K403A) of ILK KD. Figure 5D shows that the double mutation completely disrupted the parvin binding to ILK in vivo and dramatically impaired the localization of ILK to sites of FAs (Figs. 5E-5H). These data strongly demonstrate that the ILK-parvin interaction is important for the ILK localization to FAs. As a comparison, the ATP binding defective mutations did not affect the ILK localization to FAs (Figs. S5H-S5K).
The ILK binding interface on CH2 is also distinct from that for paxillin that binds the N-terminal region of CH2 (Wang et al., 2008; Lorenz et al., 2008). Overall, we have uncovered a distinct binding mode to α-parvin mediated by the pseudo-kinase active site of ILK. Importantly, the CH2 binding site in ILK also structurally rules out the possibility of α-parvin as an ILK kinase activator (Attwell et al., 2003) and explains the functional data (Figs. 3 and and4).4). Furthermore, our data provide a structural basis as to how ILK KD acts as a unique adaptor to engage α-parvin for FA assembly.
Since ILK KD was suggested to bind to integrin β CT to link integrin to focal adhesions and cytoskeleton (Hannigan et al., 1996), we asked whether the ILK KD in its tight complex with the CH2 can still bind the integrin β CTs. Remarkably, our GST-pull down assays showed that the CH2-bound ILK KD can effectively interact with both integrin β1 and β3 CTs (Figs. 6A and 6B), and α-parvin did not prevent the integrin binding to ILK (Figs. 6C and 6D). The full-length ILK in the absence of CH2 could also bind to both the integrin β1 and β3 CTs, as expected (Figs. 6E and 6F). These data indicate that the binding sites for integrin and parvin on the ILK KD do not physically overlap. Since the integrin β1 CT binding site was previously mapped to within the residues (293-451) of ILK KD (Hannigan et al., 1996), we examined the surface features of this fragment in our structure, which revealed that integrin may bind to a conserved distal hydrophobic surface of the ILK kinase C-lobe (Fig. S6). Although the precise binding site remains to be experimentally evaluated, this model explains how ILK KD acts as a distinct platform for multi-protein assembly, promoting the dynamic regulation of integrin-mediated FA assembly and the integrin-cytoskeleton linkage.
In this study, we have obtained high resolution structure of ILK KD-α-parvin CH2 complex, which provides the first definitive insight into a pseudokinase function of ILK incapable of performing the catalysis. Extensive kinase assays using both recombinant and endogenous ILK with and without putative activators (Figs. 3 and and4)4) strongly support our structure-based conclusion, but contradict with previous cell biological and biochemical data. The disagreement may arise from different experimental conditions. In particular, partially pure ILK samples used in the previous studies might have caused kinase activity via unknown kinases associated with ILK. Consistent with this possibility, we found that while partially purified ILK from the chick tissue phosphorylated MBP (Fig. 3), which was also shown before (Deng et al., 2001) (Fig. 3), it did not phosphorylate the putative physiological substrates such as integrin and Akt (Figs. 3 and and4).4). Furthermore, the phosphorylation effect was not affected at all by the addition of excess α-parvin, which would strongly inhibit the phosphorylation based on our structure (Fig. 3). Thus, great care must be taken when interpreting the results of catalytic functions derived from the partially pure samples or indirect cellular effect. Note that although we have tested the previously reported ILK substrates, it is theoretically impossible to exclude the possibility of a yet-unidentified unusual substrate for ILK, but such possibility seems unlikely because ILK does not even hydrolyze ATP - the first step of kinase reaction against any substrate.
In addition to ILK KD, three other pseudokinases (VRK3, ROP2, and STRADα) have been structurally characterized (Scheeff et al., 2009; Labesse et al., 2009; Zeqiraj et al., 2009). VRK3 and ROP2 do not bind ATP due to a substantially altered P-loop (one Gly is replaced by Met), and thus it is straightforward to classify them as pseudokinases. STRADα has several major missing catalytic features, including an altered P-loop, degenerated catalytic loop, altered DFG motif, and altered Mg binding site. These abnormal structural features are reminiscent to those in ILK. Intriguingly, the γ-phosphate of the bound ATP in STRADα is also disconnected from the degraded catalytic loop as similarly found in ILK (Figs. S7A-S7C). Such disconnection would preclude the formation of the transition state during phosphoryl-transfer even if the catalytic base was not degenerated (Cheng et al., 2005). Indeed, despite having an active conformation, STRADα was found to be catalytically inactive and also ATP was bound without hydrolysis (Zeqiraj et al., 2009). By contrast, the pseudo-active sites of STRADα and ILK have significant differences such as a shortened activation loop in ILK vs. STRADα. The two proteins are involved in completely different biological processes. A phylogenetic tree analysis indicated that STRADα and ILK may have evolved from two distinct kinase subfamilies: STRADα from STLK (Set-like kinase) whereas ILK from MLK (mixed-lineage kinase) (Manning et al., 2002).
Because of the dramatically altered catalytic motifs and the significantly short activation loop in ILK, it is less straightforward to determine precisely whether the overall structure of ILK KD mimics an active or inactive conformation of a kinase. Interestingly, using a recently developed Local Spatial Pattern protocol (Kornev et al., 2008) for examining the active or inactive conformations of kinases, we found that the overall structure of ILK KD seems to adopt a putative active conformation since it has two well-defined hydrophobic C- and R-spine motifs characteristic of active protein kinase conformations (Fig. 7). α-parvin does not seem to significantly affect this conformation since its binding site in ILK involves the conserved helices (αEF and αG) and part of P+1 loop, which are not drastically different between inactive and active kinases. Other structurally characterized pseudokinases also adopt active conformations including VRK3 and ROP2 (Scheeff et al., 2009; Labesse et al., 2009) and STRADα (in the presence of ATP and regulator MO25) (Zeqiraj et al., 2009). Preservation of active conformations of pseudokinases may be a general feature for their functions. In the case of ILK, the pseudo-active site conformation is involved in specifically recognizing α-parvin. The striking similarity of such recognition mode to the known kinase-substrate interaction (Fig. S5) suggests an interesting evolutionary consequence that pseudokinases may all use their pseudo-active sites for protein-protein interactions. The specificity of these pseudo-active site-mediated interactions may be determined by the conformations and sequences of the individual pseudo-active sites. Indeed, the G-helix orientation in ILK deviates somewhat from those of other kinase-substrate or kinase-inhibitor structures (Fig. S5G) and such deviation may be critical for the spatial specificity of ILK KD to recognize parvin. The specificity is crucial for the targeting of ILK to FAs (Fig. 5). Upon its targeting to FAs, ILK KD may further bind to other molecules such as integrin (Figs. 6 and S6) and kindlins (Tu et al., 2003) to trigger supramolecular FA assembly. What then regulates the ILK-α-parvin dissociation? ATP does not seem to play a significant role since the ILK-α-parvin complex interface is essentially identical with and without ATP. Interestingly, phosphorylation of α-parvin was shown to reduce its binding to ILK (Yang et al., 2005), suggesting that the phosphorylation may induce a conformational change of α-parvin to inhibit the CH2 binding to ILK. Although the exact mechanism remains to be determined, the α-parvin phosphorylation may provide one pathway for regulating the disassembly of ILK-mediated supramolecular complex during FA turnover.
In summary, the detailed structural and functional analysis now allows us to firmly establish ILK as a distinct pseudokinase. Our results not only resolve a longstanding controversy about the ILK kinase activity but also provide significant structural insight into how ILK regulates the integrin-cytoskeleton linkage via the integrin-ILK-parvin pathway. The conserved kinase fold but distinct pseudo-substrate active site in binding to α-parvin indicates that ILK might have evolved from an ancestral protein common to a protein kinase family but diverged during evolution to become a distinct scaffold protein. Given a crucial role of pseudokinase domain from nearly 10% of the human kinome in regulating the assembly of signaling complexes (Manning et al., 2002), our finding also paves the way for further elucidating the function of pseudokinase as a distinct protein-protein interaction module.
The bicistronic coexpression vector of the ILK KD-α-parvin CH2 complex was created by the standard subcloning strategy. Briefly, the genes encoding the human α-parvin CH2 domain (residues 248-372) (Wang et al., 2008) and the human ILK KD (residues 183-452) were amplified by PCR, and subcloned into the first and second translation cassettes of the polycistronic coexpression vector pST39 (Tan, 2001), respectively. The two variant surface cysteine residues of ILK across species were engineered to serine residues (C346S; C422S) to increase the protein solubility of the ILK KD-α-parvin CH2 complex, whereas wild type ILK (full length and KD) was used for the functional assays. A hexahistidine tag preceding a thrombin cleavage sequence was incorporated at immediately preceding the CH2 sequence. The coexpression plasmid of the ILK KD-α-parvin (full-length) complex was also created using the same strategy. The gene encoding the full-length human α-parvin was subcloned into the pET15b vector (Novagen). For coexpression of full-length ILK bound to PINCH LIM1-2, the genes encoding the human PINCH LIM1-2 (residues 1-127) and the full-length ILK (residues 1-452) were subcloned into the pST39 vector, as the above. A maltose-binding protein (MBP) gene was incorporated at the immediately preceding the PINCH LIM1-2 gene to generate the MBP-fused PINCH LIM1-2. Site-directed mutagenesis was carried out by QuikChange Site-Directed Mutagenesis Kit (Stratagene) using appropriate primer sets. All constructs were verified by the DNA sequencing analysis.
All recombinant proteins were expressed in Escherichia coli, and their purifications were performed using affinity chromatography followed by further conventional chromatography steps. Detailed procedures are described in the supplementary information.
The crystals of the ILK KD-CH2 complex (apo form) were grown at 4°C by hanging drop vapor diffusion method by mixing equal volumes of the protein solution (~0.2 mM) and a reservoir solution consisting of 0.05 M Bis-Tris Propane, pH 6.8, 12% (w/v) polyethylene glycol 5,000 mono methyl ether, 5% (v/v) 1-propyl alcohol. The co-crystals of the MgATP bound form were also grown in the similar condition as above.
Details of the data collection, structure determination and refinement are provided in the supplementary information.
The kinase assay was performed using the standard protocol from Upstate Biotechnology (see details in the supplementary information). The kinase assay was also performed by another previously reported protocol (Persad et al., 2001) and the results of these two protocols were found to be the same.
Human HeLa cells were transfected with DNA vectors encoding GFP or GFP-tagged wild type ILK or ILK mutants using Lipofectamine Plus (Invitrogen). The details of immunoprecipitation and focal adhesion localization assays are provided in supplementary information.
The binding experiments of integrin β1 CT and β3 CT to the ILK proteins (full-length or KD) were performed using the GST-pull down assays (see details in the supplementary information).
We are grateful to Dr. Peter Zwart and the staff at Lawrence Berkeley National Laboratory, and Drs. Zingwei Huang and Yongjun Zhang for technical assistance. We thank Drs. Song Tan for providing polycistronic coexpression vectors, Edward Plow, Shoukat Dedhar and other members of the Qin laboratory for useful discussions. This work was supported by grants from the NIH (J.Q. and C.W.) and AHA (S.G).
The atomic coordinates have been deposited in the Protein Data Bank (accession codes: 3KMU and 3KMW).
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