Visualization and quantification of the dynamics of protein-protein interactions in living cells can be used to explore the macromolecular events involved in signal transduction processes. In this study, functional molecular imaging using a luciferase-based complementation method demonstrated how the integrin-linked kinase (ILK)-mediated protein complex controls downstream signals. The luciferase complementation assay showed that Akt1 preferentially binds to β-parvin rather than to ILK within the complex. Moreover, photon flux from the interaction between β-parvin and Akt1 increased following serum stimulation, and the β-parvin—Akt1 interaction was dependent on phosphoinositide 3-kinase. Intriguingly, small interfering (si)RNA-mediated β-parvin knockdown increased photon flux from the interaction between ILK and Akt1, leading to stabilization of hypoxia-inducible factor-1α and increased expression of vascular endothelial growth factor-A. These data from functional molecular imaging demonstrated that β-parvin plays a regulatory role in the ILK-mediated Akt (also called protein kinase B) signaling cascades, suggesting that β-parvin might be a crucial modulator of cell survival.
Extracellular signaling is commonly mediated through mechanisms that rely on protein-protein interactions and protein phosphorylation. The dynamics of protein-protein interactions are often dependent on the activation of a particular signal transduction pathway. Even a single protein can affect various cellular functions by interacting with different partners in response to extracellular stimuli.
Recent advances in the development of molecular probes have enabled the visualization of many cellular processes and the detection of protein-protein interactions in living cells, advances typified by the fluorescence resonance energy transfer (FRET) system (Förster, 1959), in addition to complementary methods using fluorescence (Hu et al., 2002) or luminescence (Luker et al., 2004). These complementary methods designed to visualize protein-protein interactions have recently been employed in the screening of interaction partners and the potential semi-high-throughput analysis of small modulator molecules in protein complexes (Kerppola, 2006a; Kerppola, 2006b). In particular, luciferase-based complementation imaging enables sensitive real-time monitoring and quantitative analysis of protein interactions under various cellular conditions (Luker et al., 2004).
Integrins and their associated proteins mediate various intracellular signaling pathways involved in cytoskeletal remodeling and cellular processes such as cell proliferation, survival and differentiation. Integrin-linked kinase (ILK), PINCH and parvin(s) form heterotrimeric complexes that function as important regulators of integrin-mediated signaling. The complex comprising ILK, PINCH and parvins has been implicated in mediating intracellular signaling pathways through phosphorylation of the leading downstream molecule Akt (also known as protein kinase B; PKB) (Legate et al., 2006). ILK-dependent target phosphorylation is largely regulated by phosphoinositide 3-kinase (PI3K). Akt/PKB activation requires phosphorylation of Thr308 by PI3K-dependent kinase-1 (PDK1) (Alessi et al., 1997; Williams et al., 2000) and Ser473 by PDK2 [which is also known as hydrophobic motif kinase (HMK)] (Feng et al., 2004; Troussard et al., 2003). ILK is capable of controlling vascular endothelial growth factor (VEGF) transcription through Akt/PKB phosphorylation (Tan et al., 2004). ILK kinase activity is also stimulated by VEGF and other growth factors, as well as by cell adhesion to the extracellular matrix (Attwell et al., 2003).
Parvins are a family of proteins involved in linking integrins and their associated proteins with intracellular pathways that regulate actin cytoskeletal dynamics and cell survival. It includes actopaxin/CH-ILKBP/α-parvin (ParvA), affixin/β-parvin (ParvB) and γ-parvin (ParvG). ParvB localizes to focal adhesions and is involved in cell adhesion, spreading, motility and survival through interactions with its partners. ParvB accumulates and co-localizes with ILK in heart and skeletal muscle (Bendig et al., 2006; Yamaji et al., 2001). It can inhibit ILK activity and reverse some oncogenic effects in cancer cells (Mongroo et al., 2004). Furthermore, the physiological interaction of ILK with ParvB is thought to be essential in maintaining cardiac contractility (Bendig et al., 2006). Nonetheless, the precise role of ParvB and the mechanism controlling the association of ILK and ParvB remain to be delineated.
Herein, we demonstrate that luciferase complementation imaging provides a useful strategy for the instantaneous monitoring of protein-protein interactions under serum stimulation. Employment of this system enabled the detection of limited protein-protein interactions, showing that ParvB preferentially interacts with Akt1, and inhibits the interaction between ILK and Akt1 under normal cell culture conditions. ParvB knockdown using small interfering (si)RNA increased VEGF expression, with concomitant stabilized expression of hypoxia-inducible factor 1-α (HIF-1α). Based on these findings, we propose that ParvB functions as an upstream modulator of Akt/PKB through ILK, Akt/PKB and HIF-1α signaling.
Fluorescence complementation assay for protein-protein interactions involving ILK and associated proteins
It has been demonstrated that ILK protein complexes play a crucial role in regulating gene transcription and cell-cell adhesion. Molecular complementation methods were employed in an effort to detect real-time protein-protein interactions of ILK protein complexes with ParvB, PINCH1 and Akt1 (Fig. 1A). A fluorescent (or luminescent) probe was split into N-terminal and C-terminal probe fragments, each of which was attached to the N terminus or C terminus of the target protein using a linker. The optimal combination for each target protein-protein interaction leads to integration of the N-terminal and C-terminal probe fragments and light is emitted (Fig. 1A, right panel). To determine the optimal pairs of plasmids, four expression plasmids were constructed for each target protein fused with fluorescent (monomeric Kusabira-Green: mKG) or luminescent (firefly luciferase: Luc) N-terminal or C-terminal probe fragments using a linker (Fig. 1B). Protein interactions were investigated for structural conformations comprising ILK and ParvB, ILK and PINCH1, ILK and Akt1, ParvB and Akt1, and PINCH1 and Akt1. As shown in Fig. 1C, eight combinations per pair were tested to determine the optimal conformation in the living cell. The appropriate pairs of constructed plasmids were then co-transfected into 293T cells, which were then inspected using a fluorescent microscope under excitation light. One optimal pair with respect to target protein interactions was selected from among the eight combinations based on signal intensity. The optimal pairs for an mKG probe were N-ILK-linker-mKGC and N-ParvB-linker-mKGN (Fig. 1B: construct type 2 for ILK and construct type 1 for ParvB), mKGC-linker-ILK-C and mKGN-linker-PINCH1-C (construct type 4 for ILK and construct type 3 for PINCH1), N-ILK-linker-mKGN and N-Akt1-linker-mKGC (construct type 1 for ILK and construct type 2 for Akt1), N-ParvB-linker-mKGC and mKGN-linker-Akt1-C (construct type 2 for ParvB and construct type 3 for Akt1). The numbers represent the same four types of plasmids as in Fig. 1B. The plasmids encoded an unrelated protein and single plasmids did not show substantial fluorescent signals (Fig. 1D, lower six panels). Whereas the combination of ILK with ParvB or PINCH1 gave steady fluorescent signals in the cytosol (Fig. 1D, upper left panels), the combination of ParvB with Akt1 yielded stronger fluorescent signals than those yielded by the combination of ILK with Akt1 (Fig. 1D, upper right panels). The combination of PINCH1 with Akt1 did not generate substantial fluorescent signals in this assay (data not shown). In an effort to confirm the interaction between ParvB and Akt1, we performed a GST pull-down assay. Although a direct interaction between ParvB and Akt had not been reported before this study, the GST pull-down assay demonstrated that ParvB interacts directly with Akt1 (Fig. 1E). These results suggest that ParvB preferentially interacts with Akt1 and that ParvB might serve as a connecting molecule for ILK-mediated Akt/PKB signal transduction.
Luminescence complementation assay for protein-protein interactions involving ILK and associated proteins
Although a variety of methodologies have been developed for the investigation of protein interactions (Kerppola, 2006a; Kerppola, 2006b), the luciferase-based complementation assay system potentially enables straightforward and real-time quantitative analysis in living cells (Luker et al., 2004; Paulmurugan et al., 2002). As with the fluorescence complementation assay, polypeptide ends, which can adopt a broad range of intermolecular orientations, might be problematic in terms of luciferase complementation imaging (Kerppola, 2006a; Kerppola, 2006b). Optimal luciferase complementation was investigated based on results derived from fluorescence complementation assays (see Fig. 1). The optimal N- and C-terminal fragments of firefly luciferase cDNA (NLuc and CLuc, respectively) were isolated by PCR, in which NLuc and CLuc correspond to firefly luciferase amino acids 2-416 and 398-550, respectively (Luker et al., 2004). Four fusion constructs were generated for each target protein using the NLuc and CLuc cDNA fragments (see Fig. 1B). Luciferase activity was then investigated by examining target protein-protein interactions. Expression plasmid pairs were transiently transfected into 293T cells. For the control transfection, only NLuc or CLuc was employed. To determine which pair generated optimal luminescent signals, the relative luciferase activity for each target protein interaction was examined. As shown in Fig. 2A-D, the optimal pairs were as follows: NLuc-linker-ILK-C and CLuc-linker-ParvB-C (construct type 3 for ILK and construct type 4 for ParvB), CLuc-linker-ILK-C and NLuc-linker-PINCH1-C (construct type 4 for ILK and construct type 3 for PINCH1), NLuc-linker-ILK-C and N-Akt1-linker-CLuc (construct type 3 for ILK and construct type 2 for Akt1), and NLuc-linker-ParvB-C and CLuc-linker-Akt1-C (construct type 3 for ParvB and construct type 4 for Akt1). Numbers represent the same four types of plasmids as in Fig. 1B. Single plasmids fused with only NLuc or CLuc cDNA did not show substantial luminescent signals. In order to validate actual expression levels of ParvB and Akt1 protein, 2 μg of the constructed plasmid DNA was transfected into 293T cells seeded in 12-well plates. ParvB and Akt protein expression levels were analyzed by western blotting (supplementary material Fig. S1). N-terminally fused forms of Akt1 protein, NLuc-linker-Akt1-C and CLuc-linker-Akt1-C, were detected in two cleaved fragments. It has been demonstrated that Akt can be cleaved by recombinant caspase 3 at aspartic acid residues 108 and 119, resulting in the generation of a 44 kDa pleckstrin-homology (PH) domain deficient fragment (Bachelder et al., 2001; Rokudai et al., 2000). One fragment appeared to be the PH domain of Akt1 fused with a split-luciferase probe, which was detectable using anti-luciferase antibody, and the other seemed to be the kinase domain of Akt1. These data indicated that the appropriate plasmid pair exclusively provided optimal luminescent signals.
Real-time quantitative analysis of protein-protein interactions using a luciferase-based luminescent complementation assay
In an effort to determine the kinetic pattern of the ILK-ParvB and ILK-PINCH1 interactions, each optimal pair of plasmids was transfected into 293T cells in a 24-well plate. Emitted photons were measured for 30 minutes at 30 second intervals in the presence of D-luciferin using a NightOwl charge-coupled device camera. Maximal luminescence emitted from each well was observed at five to ten minutes. The photon count derived from the association of ILK with ParvB displayed a larger increase than that derived from the association of ILK with PINCH1 at the maximum bioluminescent point (Fig. 3A,B).
Although ILK has been shown to bind to Akt (Persad et al., 2001), it had not been demonstrated that ParvB is associated with Akt1. The kinetics pertaining to the interaction of Akt1 with ILK or ParvB were therefore investigated. Optimal pairs of plasmids were transfected into 293T cells under the same conditions. As shown in Fig. 3C,D, the increase in photon flux derived from the association of Akt1 with ParvB was much greater than that derived from the association of Akt1 with ILK, suggesting the preferential interaction of Akt1 with ParvB rather than ILK under living cell conditions. Maximum luminescent levels derived from the association of Akt1 with ParvB were in the range 800-1600 counts/pixels (50- to 100-fold higher than that observed for Akt1 and ILK). Kinetic analysis of Akt1-ILK complex formation showed an upward-bulging curve, with maximum luminescence levels being observed for 40-50 minutes following the addition of D-luciferin. Control expression using intact luciferase reached a peak immediately following the addition of D-luciferin (supplementary material Fig. S2). These results indicate that real-time complementary imaging can reveal characteristics unique to each target protein-protein interaction, in this case suggesting that ParvB might be an important connecting molecule in the ILK-Akt/PKB signaling pathway.
Functional complementation imaging demonstrated ParvB-Akt1 interaction following serum stimulation
In an effort to glean insight into the role of ParvB, we set out to determine whether the ParvB-Akt1 interaction was modified following serum-triggered induction. As depicted in Fig. 4A, representative pseudo-color images based on the split luciferase system revealed that photon flux derived from the interaction of ParvB with Akt1 increased under serum-rich conditions and diminished under serum-starved conditions or in the presence of a PI3K inhibitor. Consistent with these findings were the results obtained when investigating the kinetics of the ParvB-Akt1 interaction under serum-starved conditions or in the presence of a PI3K inhibitor with serum at 1-minute intervals following the addition of D-luciferin (Fig. 4B-D). The kinetic curves were logarithmic in shape. Maximum photon flux for the ParvB-Akt1 interaction for each condition was observed 20-40 minutes following serum stimulation. Serum-rich treatment increased photon flux derived from the interaction of ParvB with Akt1 (Fig. 4B). PI3K inhibitors such as LY294002 and wortmannin suppressed serum-triggered induction (Fig. 4C,D). Employment of a dual-luciferase assay also demonstrated that luciferase activity arising from the ParvB-Akt1 interaction in lysate cells was similar to photon flux in live cells (Fig. 4E). However, with respect to the ILK-Akt1 interaction and a control plasmid encoding full-length firefly luciferase, there was no significant change in luciferase activity under serum-starved or PI3K inhibitor conditions with serum (supplementary material Fig. S3A,B). These results demonstrate that serum stimulation increases photon flux derived from the interaction of ParvB with Akt1, suggesting that PI3K inhibitors attenuate the serum-stimulated interaction between ParvB and Akt1.
Interaction between ILK and Akt1 is affected by ParvB mRNA levels
Because ILK-dependent phosphorylation is regulated by PI3K, it is possible that the inhibition of PI3K activity decreases ILK activity, which subsequently impairs the phosphorylation of putative ILK substrates (Delcommenne et al., 1998). One reliable marker that relates to ILK activity concerns the phosphorylation levels of Akt/PKB (Legate et al., 2006). Given the preferential association of ParvB with Akt1, we set out to determine whether ParvB levels regulate the interaction between ILK and Akt1. NIH3T3 cells were transfected with a reporter plasmid encoding the optimal split-luciferase complementation pair ILK-ParvB (Fig. 5A) and then subjected to siRNA-mediated ParvB knockdown. The knockdown efficiency of two types of mouse ParvB siRNAs (ParvB1 and ParvB2) is shown in Fig. 5B. These siRNAs were transfected into NIH3T3 cells stably overexpressing the complementation plasmid pair ILK-ParvB (see Fig. 5A) and the luminescence photon kinetics were evaluated (Fig. 5B). ParvB knockdown using siRNA decreased photon emission associated with ILK-ParvB association (siRNA ParvB2 was able to reduce approximately 50% of the photons derived from the interaction of ParvB with ILK). We then evaluated the extent of ILK-Akt1 complex formation with loss of ParvB using both luciferase complementation imaging and a co-immunoprecipitation assay. 293T cells were transfected with the optimal complementation plasmid pair ILK-Akt1 and then subjected to ParvB knockdown using siRNA ParvB2. As depicted in Fig. 5C, we investigated the kinetics of photons derived from the interaction of ParvB with Akt1 under ParvB knockdown conditions at 1-minute intervals following serum stimulation. Interestingly, siRNA ParvB led to an increase in photon flux associated with the ILK-Akt1 interaction. To provide further evidence showing that ParvB might regulate the ILK-Akt interaction, we performed a co-immunoprecipitation assay. 293T cells were transfected with the luciferase complementation plasmid pair ILK-ParvB (Fig. 5A) and then subjected to ParvB knockdown using siRNA ParvB2 (Fig. 5B). Overexpressed ILK and ParvB were precipitated with anti-phospho-Akt (Ser473) antibodies and precipitates were analyzed by western blotting using antibodies against ILK, ParvB, total-Akt and phospho-Akt (Ser473). As shown in Fig. 5D, decreased ParvB protein levels resulted in increased interaction between ILK and phospho-Akt (Ser473). These results suggest that ParvB might act as a potential modulator of the signal transduction associated with the ILK-Akt/PKB complex.
ParvB knockdown increased HIF-1 and VEGF-A expression levels
We showed above that ParvB protein levels regulate the ILK-Akt1 interaction following serum stimulation. It has been demonstrated that HIF-1α is an important downstream effector that acts through the PI3K-Akt/PKB signaling pathway (Jiang et al., 2001). Furthermore, HIF-1α can regulate the expression of VEGF (Forsythe et al., 1996; Liu et al., 1995). In an effort to examine the effect of ParvB downregulation on downstream targets of the ILK-Akt signaling pathway, HIF-1α and VEGF protein expression levels were investigated following siRNA-mediated ParvB knockdown. ParvB is ubiquitously expressed, but enriched in heart and skeletal muscle (Bendig et al., 2006; Sepulveda and Wu, 2006). We transfected rat ParvB-specific siRNA into rat cardiomyocytes and confirmed the knockdown effect of ParvB (Fig. 6A). Western blot analysis demonstrated increased expression levels of endogenous HIF-1α and VEGF-A with ParvB knockdown in rat cardiomyocytes. HIF-1α and VEGF-A expression levels were quantified and statistically analyzed (Fig. 6B,C).
In an effort to further investigate the increase in HIF-1α expression induced by siRNA ParvB, another reporter plasmid was employed containing oxygen-dependent degradation (ODD)-luciferase, which comprises the ODD domain of HIF-1α fused to luciferase. Because the stability of HIF-1α is tightly regulated through the ODD domain (Harada et al., 2002), luciferase activity associated with the fusion protein reflects stabilized HIF-1α levels. ODD-luciferase cDNA was subcloned into the pcDNA3.1 expression plasmid (Fig. 6D) and transfected into NIH3T3 cells. ODD-luciferase-expressing stable transformants were obtained following G418 selection. We then introduced siRNA ParvB1 or ParvB2 into the ODD-luciferase-expressing NIH3T3 transformants. As shown in Fig. 6E, ParvB knockdown resulted in a marked increase in the expression of ODD-luciferase. Moreover, the hypoxia mimetics CoCl2 and desferrioxamine (DFO) also increased ODD-luciferase-derived photon flux in the NIH3T3 transformants (Fig. 6F). To further confirm the requirement for ILK in the induction of HIF-1α stability under loss of ParvB, both siRNA ParvB1 and siRNA ILK were introduced into ODD-luciferase-expressing NIH3T3 transformants. As shown in Fig. 6G, knockdown using both ParvB and ILK did not increase ODD-luciferase-derived photon flux in the NIH3T3 transformants (Fig. 6G).
Taken together, these results indicate that decreased levels of ParvB stabilize HIF-1α in the presence of ILK, suggesting that ParvB downregulation might mimic hypoxic conditions through the ILK-Akt/PKB signaling pathway.
Complementation strategies using an imaging probe with appropriate protein reconstitution enable visualization of steady-state complexes formed between protein pairs. One of the benefits associated with the use of these techniques is the exclusion of certain secondary effects or potential artifacts caused by cell lysis (Kerppola, 2006a; Kerppola, 2006b). Of the available complementation strategies, luciferase-based luminescence complementation imaging can be employed as a facile and broadly applicable approach (Luker et al., 2004). In this study, we demonstrated two complementary methods employing fluorescent or luminescent protein probes. Both imaging methods required that eight combinations of reporter plasmids be examined to determine optimal signal gain from the target protein-protein interaction. Although fluorescence complementation enabled visual observation of the actual protein interactions, it might not necessarily allow comparison of protein-protein interactions in living cells under the microscope. By contrast, luminescence complementation enables comparison of even weak protein interactions under almost real-time conditions by quantifying luminescent signals with a high signal-to-noise ratio. Furthermore, employment of the luciferase complementation strategy allows the observation of rapid changes in target protein interactions in subcellular compartments under various conditions. In fact, our extensive complementary studies demonstrated that ParvB preferentially binds to Akt1 rather than ILK under living cell conditions. Moreover, luciferase complementation imaging revealed that real-time changes in photon-based kinetics associated with the ParvB-Akt1 interaction were consistent with results obtained following the use of extracellular stimuli, such as growth factors and associated signal inhibitors. Thus, employing the complementation strategy with appropriate probes could provide unique findings that reflect real-time cellular responses to external stimuli. Based on the current studies, employment of functional molecular imaging using complementary methods promises to be a beneficial strategy for the exploration of molecular mechanisms pertaining to signal transduction.
The ILK-associated protein complex is profoundly involved in altering the flux of the PI3K-Akt/PKB signaling pathway (see Fig. 7). ILK-dependent phosphorylation is regulated in a PI3K-dependent manner. PI3K inhibitors reduce ILK activity and impair the phosphorylation of putative ILK substrates in cell culture (Delcommenne et al., 1998). ParvB is a binding partner of ILK and the CH2 domain of ParvB is phosphorylated by ILK. These findings suggest that ParvB probably plays a role in actin cytoskeleton remodeling and cell spreading with its binding partners, α-actinin and αPIX (Mongroo et al., 2004; Yamaji et al., 2004; Yamaji et al., 2001). One new finding in our study showed that the real-time photon-based kinetics of the ParvB-Akt interaction correlated with results obtained following the use of extracellular stimuli, such as the presence of serum with or without PI3K inhibitors (Fig. 4). Moreover, decreased levels of ParvB protein were associated with a marked increase in photons derived from the ILK-Akt interaction (Fig. 5C,D). Although the interaction between ParvB and Akt had not been reported before this study, ParvB might cooperate with Akt and play a regulatory role in ILK-Akt complex formation (Fig. 7).
The role of ParvB in relation to Akt/PKB downstream effectors had not previously been sufficiently addressed. In this study, we demonstrated that siRNA-mediated ParvB knockdown increased HIF-1α and VEGF expression in rat cardiomyocytes (Fig. 6B,C). In particular, we showed that the phenomenon was correlated with HIF-1α stabilization. In our luciferase-based real-time imaging, ODD-luciferase expression levels increased with siRNA-mediated ParvB knockdown, firmly supporting the notion of HIF-1α stabilization (Fig. 6E). Furthermore, the hypoxia mimetics CoCl2 (100 μM) and DFO (20 μM) increased luminescent signals in ODD-luciferase-overexpressing NIH3T3 cells. The effect of ParvB knockdown was the same as that of hypoxia mimetic agents. However, knockdown using both ParvB and ILK siRNAs did not increase luminescent signals in ODD-luciferase-overexpressing NIH3T3 cells (Fig. 6G). These results suggest that ParvB expression levels potentially control HIF-1α levels in the presence of ILK and that HIF-1α levels subsequently alter the expression of target genes such as VEGF-A. Taken together with the results presented here, one possible scenario can be outlined (Fig. 7): growth factors trigger ILK-Akt/PKB signaling activation and consequently ParvB could be phosphorylated by activated ILK, leading to increased interaction between ParvB and Akt. In the normal and physiological state, ParvB might act as a modulator to prevent the transmission of excess signals, such as those derived from growth factors, in the ILK-Akt/PKB signaling cascade (Fig. 7A). However, without regulation of ParvB, excess signals might be transmitted in an uncontrolled manner to downstream targets such as HIF1-α or VEGF (Fig. 7B). The notion of ParvB acting as a modulator might be supported by the finding that ParvB is downregulated in breast cancer (Mongroo et al., 2004).
With respect to the role of ParvB in ILK—Akt/PKB—HIF-1α signaling, it has been demonstrated that loss of ParvB expression upregulates ILK activity in tumors (Mongroo et al., 2004) and that ILK activation triggers a cascade of Akt/PKB phosphorylation events in tumor cells (Majumder et al., 2004). Because it has been reported that transcriptional responses to Akt/PKB activation are involved in the mTOR-dependent regulation of HIF-1α in an Akt-driven prostate intraepithelial tumor, ParvB might be involved in HIF-1α stabilization via ILK-Akt/PKB signaling in tumor cells. Thus, it is believed that our results are consistent with the role of ParvB in tumor cells.
Although further investigations might be necessary given these findings, employment of the complementary protein imaging strategy enabled the visualization and quantification of protein-protein interactions. This approach allowed the detection of real-time alterations in ILK-Akt signaling under conditions involving extracellular stimulation of living cells. These compelling data demonstrate that ParvB plays a regulatory role in ILK-Akt/PKB-HIFα signaling.
Materials and Methods
cDNAs and plasmid construction
For the isolation of ILK, ParvB, PINCH1 and Akt1 cDNA (GenBank accession numbers: NP_034692, NM_133167, NM_026148 and NM_009652, respectively), total RNA was extracted from the heart of BALB/c mice using ISOGEN (Nippon Gene, Tokyo, Japan) following the manufacturer's instructions. 1 μg of total RNA was reverse transcribed using random hexanucleotide primers and SuperScript III (Invitrogen Carlsbad, CA) according to the manufacturer's instructions. The entire coding sequence of these cDNAs was amplified by PCR using appropriate cloning primers (supplementary material Table S1). PCR was performed using EX Taq polymerase (Takara Bio, Otsu, Japan) as follows: 94°C for 2 minutes, 59°C for 1 minute, 72°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 1 minute, with a final extension step at 72°C for 5 minutes. PCR products were cloned into 2.1 TOPO vector (Invitrogen, Carlsbad, CA). To detect KG fluorescent signals formed by the protein-protein interactions, ILK, ParvB, PINCH1 and Akt1 cDNAs were subcloned into phmKGN-MC, phmKGC-MC, phmKGN-MN and phmKGC-MN plasmids of the Fluo-chase kit (Medical & Biological Laboratories, Woburn, MA), which included the Kozak consensus sequence.
For the split firefly luciferase complementation assay, optimal pairs of N- and C-terminal cDNA fragments were amplified by PCR from pGL4.10 plasmid DNA (Promega, Madison, WI). The N- or C-terminal cDNA fragment was inserted into the pcDNA3.1 expression vector (Invitrogen). ILK, ParvB, PINCH1 and Akt1 cDNAs were fused in-frame to the N-terminal luciferase fragment (NLuc) or the C-terminal luciferase fragment (CLuc) containing a linker sequence.
Both ParvB cDNA fragments comprising N-terminal CLuc (CLuc-linker-ParvB) and ILK with N-terminal NLuc (NLuc-linker-ILK) were inserted into the pILES2-EGFP expression vector (Clontech Laboratories, Palo Alto, CA). An internal ribosomal entry site (IRES) sequence was inserted upstream of the start site of the NLuc-linker-ILK cDNA.
All PCR primer sequences for cDNA cloning and subcloning are shown in supplementary material Table S1.
Cell culture and transfection
NIH3T3 and 293T cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma Chemical Co., St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, 10 mM HEPES buffer, 2 mM glutamine, 100 units/ml penicillin G and 100 μg/ml streptomycin sulfate. Hearts from Wistar rat neonates were removed under a microscope, minced and digested four times at 37°C using a mixture of 1 mg/ml collagenase (Worthington Biochemical Co, Lakewood, NJ) and Hank's solution. Cardiac cells were neutralized and washed by low-speed centrifugation in 10% FCS-DMEM. After enzymatic digestion of their tissues, a cardiomyocyte fraction was prepared by Percoll gradient centrifugation (GE Healthcare). Cardiomyocytes were plated on wells pre-incubated with FCS.
Transient transfection was performed with an 80% confluent cell culture of 293T and NIH3T3 cells using Lipofectamine 2000 or Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions. For serum stimulation, transfected cells were serum starved in 1% FCS for 18-20 hours and followed by FCS addition immediately before the assay. For PI3K inhibition, cells were pretreated with LY294002 (Sigma) or wortmannin (Sigma) at the concentration specified in the figure legends for 2 hours before the assay.
For the isolation of stable transformants that express the optimal complementation pair ILK and ParvB with EGFP (see Fig. 5A) and ODD-luciferase (Harada et al., 2002) (see Fig. 6D), NIH3T3 cells were grown in 10% FCS-DMEM containing 1000 μg/ml G418 at 2 days post-transfection. Following 21 days of G418 selection, neomycin-resistant colonies were isolated.
For the siRNA experiments, ParvB-specific siRNA duplexes, ILK-specific siRNA duplexes and an unrelated siRNA duplex as a control were purchased from Invitrogen. The siRNA transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The final siRNA concentration was 20-40 nM.
Complementation imaging assay
The fluorescence complementation assay was performed using the Fluo-chase Kit (Medical & Biological Laboratories). 293T cells were co-transfected using the split-mKG vector system according to the manufacturer's instructions. Cells were inspected and photographed at 36-48 hours post-transfection using a fluorescent microscope equipped with a UV light source (excitation 470-495 nm; emission 510-550 nm).
For the luciferase-based complementation imaging, firefly luciferase cDNA was split into two fragments. 293T cells were co-transfected with the appropriate eight pairs of constructed plasmids (see Fig. 1C). Cells were plated and then transiently co-transfected with the appropriate pairs of plasmids using Lipofectamine 2000 (Invitrogen). At 36-40 hours after transfection, photon flux from the cells was measured using the NightOWL imager at 1-minute intervals following the addition of D-luciferin (potassium salt; Biosynth, Postfach, Switzerland). Cells were also assayed at 36-40 hours following transfection using the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.
Immunoprecipitation and western blotting
293T cells were transfected with the constructed plasmid encoding the optimal luciferase complementation plasmid ILK-ParvB. Cells were lysed with a lysis buffer (1% Triton X in 50 mM HEPES, pH 7.4, containing 150 mM NaCl, 1 mM Na3VO4, 50 mM NaF and protease inhibitors). Cell lysates were incubated overnight at 4°C with anti-phospho-Akt (Ser473) antibodies (Cell Signaling Technology, Beverly, MA) and then mixed with protein-G agarose beads (GE Healthcare, Piscataway, NJ). Precipitates were washed four times and then subjected to western blotting.
For western blot analysis, cells were lysed 2-3 days following transfection with a lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and protease inhibitors). Cell lysates were subjected to 5-20% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Blots were blocked using 2% bovine serum albumin (Sigma) or 5% ECL blocking agent (GE Healthcare) in TBST buffer (20 mM Tris-HCl, 150 mM NaCl and 0.1% Tween 20). Membranes were then incubated overnight at 4°C with target primary antibodies in the blocking buffer at a dilution recommended by the manufacturer [phospho-Akt (Ser473), Akt and ILK: Cell Signaling Technology, Beverly, MA; HIF-1α: Chemicon, Temecula, CA; VEGF-A: Immuno-Biological Laboratories Co, Gunma, Japan; luciferase: Promega; ParvB and GAPDH: Santa Cruz Biotechnology Inc, Santa Cruz, CA]. Membranes were washed extensively in TBST buffer and then incubated with Clean-Blot IP Detection Reagent HRP (Thermo Fisher Scientific Inc., Rockford, IL), anti-rabbit IgG (Cell Signaling Technology), anti-goat IgG (Santa Cruz Biotechnology) or protein A (Bio-Rad, Hercules, CA) for 1 hour in TBST buffer. Membranes were finally washed with TBST buffer and signals were visualized using ECL plus (GE Healthcare).
Expression and purification of recombinant proteins
The ParvB fragment generated by PCR was cloned between the BamHI and EcoRI restriction sites of pGEX-6P-1 (GE Healthcare). GST-ParvB and GST were expressed in Escherichia coli BL21 (DE3) and purified using GST Bind Kits (Novagen, Darmstadt, Germany) according to the manufacturer's instructions.
GST pull-down assay
For the pull-down assay, 5 μg of either GST or GST-ParvB mixed with 1 μg His-tagged Akt1 (Invitrogen) were bound to glutathione-coated agarose beads in 500 μl of binding buffer (150 mM NaCl, 50 mM HEPES, 0.1% NP-40, pH 7.4, 1 mM dithiotreitol) for 2 hours at 4°C. Bound proteins were washed three times and eluted with SDS sample buffer, separated by SDS-PAGE, and detected by western blotting and Coomassie brilliant blue (CBB) staining.
Quantitative real-time RT-PCR
Total RNA was isolated using ISOGEN (Nippon Gene Co.) according to the manufacturer's instructions. 1 μg of total RNA was reverse transcribed with oligo-dT primers using SuperScript III (Invitrogen) according to the manufacturer's instructions. ParvB gene expression was quantified using iQ SYBR Green Supermix (Bio-Rad). Each assay was performed in triplicate. ParvB transcript levels were determined as expression levels relative to 18S rRNA. The following primers were used: rat ParvB, 5′-TTGTCCCCCTTCACAACTTC-3′ and 5′-GTGTAAAGGACCCGCAGAGT-3′; 18S rRNA, 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′. Each experiment was performed three times with similar results.
Comparisons between multiple groups were performed using ANOVA followed by post hoc tests or Student's t-test. A P value less than 0.05 was considered significant.
We thank J. Hotta, Y. Ohide and M. Kumagai for excellent technical assistance. This study was supported in part by a grant to T.M. from the Health and Labour Science Research Grants (Research on Biological Resources), a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS; project number 20591326; 2008), and a grant from the Strategic Research Platform for Private Universities: matching fund subsidy from MEXT (2008-), and a Research Award to JMU Graduate Student (M.K.).