Regulation of receptor for activated C kinase 1 protein by the von Hippel–Lindau tumor suppressor in IGF-I-induced renal carcinoma cell invasiveness

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von Hippel–Lindau (VHL) tumor suppressor loss is associated with renal cell carcinoma (RCC) pathogenesis. Meanwhile, aberrant activation of the insulin-like growth factor-I (IGF-I) signaling has been implicated in the development of highly invasive metastatic RCC. However, the link between VHL inactivation and RCC invasiveness is still unexplored. Here, we show that the receptor for activated C kinase 1 (RACK1) is a novel pVHL-interacting protein. pVHL competes with IGF-I receptor (IGF-IR) for binding to RACK1 thus potentially modulating the downstream IGF-I signal pathway. Upon IGF-I stimulation, pVHL-deficient RCC cells exhibit increased RACK1/IGF-IR binding and increased IGF-IR tyrosine kinase activity. pVHL-deficient RCC cells also demonstrate elevated PI3K/Akt signaling and matrix metalloproteinase-2 activity that culminates in enhanced cellular invasiveness, which can be partially suppressed by RACK1 small interfering RNA. Domain mapping analysis showed that the pVHL α-domain and the RACK1 WD 6–7 domains are critical for the interaction. Additionally, the RACK1 expression level is not regulated by pVHL expression status, suggesting that pVHL modifies RACK1 functions independent of the VHL/elongin E3 ubiquitin ligase complex. Our data indicate that RACK1 serves as a direct mediator between loss of pVHL function and enhanced IGF-IR signaling pathway in RCC.


Renal cell carcinoma (RCC) accounts for 2% of all adult malignancies and constitutes 85% of all primary renal tumors (Jemal et al., 2005). One salient characteristic of RCC is its marked propensity for invasiveness. At the time of diagnosis, 25% of RCC patients have metastatic lesions and generally have a very poor prognosis (Motzer and Russo, 2000; Cohen and McGovern, 2005). Therefore, identifications of molecules involved in RCC invasive behavior will provide useful information on efficiently treating RCC metastases. The process of tumor cell metastasis is partially regulated by insulin-like growth factor-I (IGF-I), which is a potent mediator of tumor cell migration and invasion (Tanno et al., 2001). IGF-I and its receptor IGF-IR have a major role in the modulation of cellular behaviors in a variety of human tumors including renal cancers (Dupont et al., 2003). Increased activation of IGF-I signaling pathway has been implicated in many animal and human malignancies (Macaulay, 1992). Ligand-dependent activation of the IGF-I receptor tyrosine kinase results in a cascade of intracellular tyrosine phosphorylations and triggers multiple downstream signals including the PI3K/Akt pathway implicated in malignant cellular invasiveness. The constitutive activation of IGF-IR always culminates in the initiation of cell transformation and invasion. In several animal models, the IGF-I/IGF-IR pathway has been identified as a major promoter of tumor invasion and metastases (Brodt et al., 2000). Also, blockage of its downstream signals by IGF-IR antibodies results in suppression of RCC cell growth (Rosendahl and Forsberg, 2004; Schips et al., 2004). Therefore, defining the mechanisms of IGF-IR activation in RCC cells, and how its signaling stimulates invasiveness, will provide a better understanding of RCC biology.

Inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene is responsible for VHL disease, which is a dominantly inherited familiar syndrome characterized by the development of a variety of benign and malignant tumors including RCC. Though inherited VHL disease accounts for only 2% of all kidney cancer cases, somatic VHL mutations occurs in the majority of sporadic RCC (Foster et al., 1994; Gnarra et al., 1994). The VHL gene product (pVHL) is the substrate recognition component of an E3 ubiquitin ligase complex (VCB–CUL2) that also contains elongin B, elongin C and cullin 2 proteins (Kibel et al., 1995; Pause et al., 1997). The VCB–CUL2 complex targets hypoxia-inducible factor (HIF)-1α and -2α for oxygen-dependent degradation and regulates cellular response to hypoxia (Maxwell et al., 1999; Ohh et al., 2000). Meanwhile, there is evidence that pVHL possesses tumor suppressor activities unrelated to its regulation on HIF-α, such as regulation of mRNA transcription, mRNA stability (Gnarra et al., 1996; Na et al., 2003) and extracellular matrix assembly (Ohh et al., 1998).

Receptor for activated C kinase 1 (RACK1) is the first member identified in the family of RACK (Mochly-Rosen et al., 1995). RACK1 also belongs to the Trp-Asp 40 (WD40) repeat family. It contains seven highly conserved internal consensus WD40 repeats, which are predicted to form a seven-bladed β-propeller structure (Steele et al., 2001). RACK1 has been found to interact with protein kinase C, Src kinase, Jun-N-terminal kinase, and several transmembrane receptors such as integrin β (Liliental and Chang, 1998; Rodriguez et al., 1999). Therefore, RACK1 may have a pivotal role in many critical biological responses by serving as a mediator that integrates different signaling pathways (McCahill et al., 2002).

In this study, we identified a novel interaction between pVHL and RACK1. We also found pVHL competes with IGF-IR for binding to RACK1. We show that upon IGF-I treatment, pVHL-deficient RCC cells exhibit increased IGF-IR-RACK1 interaction, and elevated IGF-IR, Akt activation. Introducing RACK1 small interfering RNA (siRNA) into these cells can reverse these effects. pVHL-deficient RCC cells also demonstrate elevated IGF-I-induced matrix metalloproteinase (MMP)-2 expression and activation that can be effectively suppressed by RACK1 siRNA and LY294002. Knockdown of RACK1 expression in these pVHL-defective RCC cells also results in the suppression of IGF-I-induced RCC cellular invasiveness. Taken together, these results indicate the pVHL-RACK1 interaction negatively regulates the activation of the IGF-IR/Akt/MMP-2 signals and suppresses IGF-I-dependent invasiveness in RCC cells.


pVHL interacts with RACK1

Recent studies have shown that pVHL associates with many cellular proteins and appears to have biological functions unrelated to its ability to regulate HIF-α levels(Hansen et al., 2002; Zhou et al., 2004; Alberghini et al., 2005; Zagzag et al., 2005). To address novel tumor suppressor functions of pVHL, we performed several rounds of yeast two-hybrid screening to identify pVHL-interacting proteins. The ‘bait’ used in these screens was the full-length pVHL cloned into the GAL4 DNA-BD/bait vector. The pretransformed human kidney complementary DNA (cDNA) library was employed as the ‘prey’. Among 21 positive candidate clones from 2.5 × 107 mating diploids screened, one clone encoding human RACK1 was identified.

The interaction between pVHL and RACK1 was confirmed both in vitro and in vivo. To test the direct binding of pVHL and RACK1, we carried out an in vitro GST pull-down assay (Figure 1a). GST-VHL was incubated with methionine-labelled in vitro-translated RACK1. The results showed that pVHL specifically associated with RACK1 in vitro, whereas the control GST vector did not. To further confirm the interaction in vivo, we conducted co-immunoprecipitation (IP) experiments using human kidney 293T cell lysates. FLAG-tagged pVHL (FLAG-pVHL) was transiently transfected with HA-tagged RACK1 (HA-RACK1) or the control HA vector before co-IP. The results revealed that FLAG-pVHL co-immunoprecipitated with HA-RACK1, but not the mock vector (Figure 1b). We also conducted IP of the endogenous pVHL and RACK1 in 786-O and RCC4 renal cancer cell lines stably expressing HA-VHL or the mock HA vector (Figures 1c and d). In both cell lines, we found pVHL robustly bound to endogenous RACK1, which confirmed the interaction between pVHL and RACK1 in vivo. Using double immunofluorescence staining, we next examined whether the association between pVHL and RACK1 resulted in co-localization in human kidney tissues (Figure 1e). As shown in Figure 1e, pVHL and RACK1 were both present mainly in the renal tubular epithelial cells. Moreover, both pVHL and RACK1 co-localized to the cytoplasm, where they accumulated as relatively large aggregates.

Figure 1

pVHL interacts with RACK1. (a) pVHL interacts with RACK1 in vitro. GST pull-down assays with in vitro translated RACK1. (b) pVHL interacts with RACK1 in HEK293T cells. (c and d) Endogenous RACK1 and pVHL interactions in RCC cells. (e) Representative immunofluorescence staining of pVHL colocalizes with RACK1 in clinical human tissues. pVHL colocalizes with RACK1 to the cytoplasm of tubular epithelial cells in human renal tissue. Endogenous pVHL and RACK1 were detected by immuno-fluorescence using anti-pVHL and anti-RACK1 antibodies and visualized using rhodamine- and fluorescein-labeled secondary antibodies, respectively. The images were overlapped (merge) to reveal colocalization.

We then mapped the regions on RACK1 required for the interaction. Three RACK1 deletion mutations WD1–4, WD5–7 and WD6–7 were generated (Figures 2a and b). We observed that all mutants containing WD6–7 interacted with co-transfected FLAG-VHL in vivo. In contrast, WD1–4 seemed to be dispensable for the association (Figure 2c). To define the binding domain on pVHL for RACK1, we also generated several GFP-tagged truncated pVHL fusions and examined their ability to bind RACK1 in vivo. Co-IP assays showed that all fusions containing α-domain (amino acids 155–192) bound to co-transfected HA-RACK1 in 293T cells, indicating that the α-domain of pVHL is critical for the pVHL-RACK1 interaction (Figures 2d and e).

Figure 2

Domains required for interaction between RACK1 and pVHL. (a) Schematic depictions of RACK1 wild type (wt) and its truncation mutants used in the experiments. The seven WD domains of RACK1 and the corresponding amino acids are indicated. (b) Amino acid sequence alignment of the seven WD domains in human RACK1. Conserved amino acids among these WD domains are shaded. Note that WD6–7 domains are more diverged from WD1–5. (c) pVHL interacts with WD6–7. HEK293T cells were transiently co-transfected with plasmids encoding FLAG-pVHL and three HA-tagged truncation mutants of RACK1, WD1–4, WD5–7 or WD6–7. Cell lysates were immunoprecipitated (IP) by anti-FLAG antibody followed by immunoblotting (IB) with anti-HA. (d) Schematic structure diagrams of pVHL and its truncation mutants we applied in the domain mapping experiments. (e) RACK1 binds to the α-domain of pVHL. HEK293T cells were transiently co-transfected with plasmids encoding HA-RACK1 and six GFP-tagged truncation mutants of pVHL. Only mutants containing aa 155–192 (α domain) formed complexes with RACK1.

RACK1 is not a target of pVHL-mediated proteasomal degradation

Given the oxygen-sensing role of pVHL in HIF-α degradation, we tested whether RACK1 protein expression is regulated by pVHL or oxygen deprivation. First, we measured RACK1 expression levels in a series of renal cancer cell lines with different pVHL statuses. We found levels of RACK1 protein expression were pVHL status-independent (Figure 3A). We then tested whether RACK1 expression was regulated by oxygen concentration and subjected to proteasomal degradation (Figure 3B). Our results showed that RACK1 levels in normoxia and hypoxia were comparable. Addition of 10 μM MG132, a potent proteasome degradation inhibitor, caused no major effect on RACK1 expression either. All these results suggest that, unlike HIF-1α and -2α, RACK1 is not a target of pVHL-mediated proteasomal degradation. Meanwhile, increased RACK1 expression was not able to disrupt the interaction between pVHL and elongin B, C in HEK293 cells, suggesting that RACK1 do not compete with elongin B, C for pVHL interaction (Figure 3C).

Figure 3

RACK1 is not a target of pVHL-mediated proteasomal degradation and neither regulated by hypoxia. (A) Protein levels of RACK1 in several renal cancer cell lines with wild-type (WT) or mutant (MU) pVHL were measured. (B) Hypoxia does not affect basal expression levels of RACK in cells with different pVHL statuses. Western blot results showed levels of RACK1 in cells cultured in normoxia and cells treated with hypoxia or MG132. β-actin expressions were shown as a protein loading control. (C) Increased RACK1 expression does not disrupt the VBC–deubiquitination complex formation in HEK293 cells. (D) Representative expression of pVHL and RACK1 in primary and metastatic human RCC tissues. Normal pVHL expression in a non-neoplastic tissues (a, c) and dramatically diminished pVHL expression in RCC tumor tissues (b, d). Regardless of pVHL expression levels, there is no obvious change in RACK expression in most tumor tissues (e, f). Some metastatic tissues demonstrated increased RACK1 expression (g, h). IB, immunoblotting.

Next, we investigated pVHL and RACK1 protein expressions in clinical RCC tissues. Formalin-fixed and paraffin-embedded specimens of clear cell primary (n=12) and metastatic (n=4) RCCs were collected for IHC analysis. As seen on Figure 3D, non-neoplastic renal tissue showed strong pVHL immunoreactivity in ductal epithelium. In the primary RCC group, pVHL expression was significantly diminished or completely lost in 9 of 12 (75%) samples. In the metastatic RCC group, all four samples (100%) completely lost pVHL expression. Moreover, all normal or tumor tissues express fair amount of RACK1 and no correlation between pVHL and RACK1 immuno-reactivity was found. Among 9 of 13 (70%) tumor samples that demonstrate diminished or lost pVHL expression, there is no significant change of RACK1 expression (Figure 3D). One primary clear cell RCCs showed lowered RACK1 expression; two metastatic clear cell RCCs showed higher RACK1 expression.

RACK1 contributes to enhanced IGF-IR kinase activity in pVHL-deficient RCC cells

Several reports on RACK1 suggest a role in mediating IGF-IR signals (O'Connor, 2003). Meanwhile, overexpression of pVHL in renal cancer cells has been shown to inhibit IGF-IR signaling (Datta et al., 2000). Whether pVHL status affects IGF-IR kinase activity and whether this effect is RACK1-dependent are questions important to our understanding of how pVHL regulates RCC progression. To address these questions, in vitro kinase assays were performed after IP of IGF-IR from both pVHL-positive and pVHL-negative RCC cell lysates. The IGF-IR kinase activities were determined by measuring the amount of ATP incorporated into the peptide substrate poly (Glu-Tyr; Figure 4a). Our results showed that, in an unstimulated state, basal IGF-IR tyrosine kinase activity was almost the same in both pVHL-deficient and pVHL-positive RCC cells. However, after IGF-I stimulation for 30 min, pVHL-deficient RCC cells exhibited a much greater (85%) increase in IGF-IR kinase activity compared with pVHL-positive cells. These data suggest that loss of pVHL in RCC cells causes a marked increase of IGF-IR tyrosine kinase activity upon IGF-I stimulation.

Figure 4

RACK1 contributes to the enhanced IGF-IR kinase activity seen in pVHL-deficient cells. (a) Enhanced in vitro IGF-IR kinase activity in pVHL-null RCC cells. The in vitro kinase activity was counted by liquid scintillation counting. The western blot insert shows the levels of IGF-IR in the aliquot of each immunoprecipitated sample. (b) Elevated IGF-IR substrate IRS-2 phosphorylation in pVHL-deficient RCC cells. (c) Endogenous VHL in HEK293 cells was knocked down using VHL-specific siRNA. Both the in vitro and in vivo IGF-IR kinase activity was measured. (d) RACK1 siRNA dramatically suppressed IGF-IR kinase activation in pVHL-deficient RCC cells. The IGF-IR kinase activity was measured by the in vitro substrate phosphorylation assay as described in (a). The insert shows the levels of RACK1 and pVHL expression in representative samples and the effectiveness of the siRNA knockdown method. IB, immunoblotting.

To investigate the effect of pVHL on IGF-IR kinase activation in vivo, we also compared phosphorylation of IGF-IR substrates. One well-documented IGF-IR substrate, IRS-2, was immunoprecipitated from RCC cells with different pVHL statuses and its IGF-I-induced phosphorylation was examined by western blot with anti-phosphotyrosine antibody 4G10. Our results showed that IGF-I-induced IRS-2 phosphorylation was dramatically increased in pVHL-deficient cells when compared with the pVHL-positive cell lines (Figure 4b). In addition, siRNA-mediated knockdown of endogenous VHL in HEK293 embryonic kidney cells resulted in similarly marked upregulation of IGF-IR kinase activity and IRS-2 phosphorylation upon IGF-I stimulation (Figure 4c).

To evaluate the role of RACK1 in pVHL-mediated suppression of IGF-IR kinase activity, we generated a siRNA pool for RACK1. Transient transfection of RACK1 siRNA in RCC cells led to dramatic suppression of RACK1 expression. When RACK1 siRNA was applied to pVHL-deficient RCC cells, IGF-I-induced IGF-IR kinase activation was reduced by more than 60% after 30 min of IGF-I stimulation. However, in pVHL-positive cells, transient knockdown of RACK1 only slightly inhibited the kinase activity by 5% (Figure 4d). Taken together, these data suggested a critical role for RACK1 in enhancing IGF-IR kinase activity seen in pVHL-deficient RCC cells.

pVHL-deficient RCC cells show enhanced binding between RACK1 and IGF-IR

RACK1 was reported to bind to IGF-IR and regulate its downstream signaling. Thus, to investigate whether pVHL had an impact on the interaction between RACK1 and IGF-IR, RCC cells with different pVHL expression statuses were serum starved and treated with IGF-I. Anti-IGF-IR immunocomplexes from these cells were separated by SDS–polyacrylamide gel electrophoresis and immunoblotted with anti-RACK1 to compare any interaction changes (Figure 5a). Our results showed that stimulation of RCC cells with IGF-I caused increased association between RACK1 and IGF-IR. pVHL-deficient RCC cells demonstrated greater amount of RACK1 co-immunoprecipitated with IGF-IR compared with pVHL-positive RCC cells. We also examined the association of RACK1 with pVHL in response to IGF-I in human kidney 293T cells. 293 cells were transfected with FLAG-RACK1 and HA-VHL, and then treated with IGF-I. The association of RACK1 with pVHL was examined by IP with anti-HA followed by western blot with anti-FLAG. The results showed that the association of RACK1 with pVHL was enhanced by IGF-I (Figure 5b). These data suggest that pVHL expression status affect the interaction of RACK1 with IGF-IR. Thus, in RCC cells lacking a functional pVHL, more RACK1 is able to bind with IGF-IR and potentially regulates its kinase activity.

Figure 5

pVHL disrupts the association of IGF-IR and RACK1. (a) RCC cells stably expressing HA-pVHL (pVHL+) or mock HA vector (pVHL-) were serum-starved and treated with IGF-I. Cell lysates were immunoprecipitated (IP) with anti-IGF-IR antibody. The immunoprecipitates were fractionated by SDS–polyacrylamide gel electrophoresis and detected by western blot. (b) IGF-I induces enhanced association of pVHL with RACK1. 293 cells were either untreated or treated with IGF-I. Cell lysates were immunoprecipitated with anti-HA followed by western blot with anti-FLAG. (c) pVHL dissociates RACK1 from IGF-IR. 293 cells were transfected with expression plasmids for HA-tagged IGF-IR (1 μg) with various amounts of HA-pVHL DNA (0.1, 0.25, 0.5, 0.75 and 1 μg). Association of pVHL and IGF-IR with endogenous RACK1 was determined by IP with anti-RACK1 followed by western blot with anti-HA. (d) pVHL does not bind to IGF-IR. Cell lysate expressing RACK1 and IGF-IR was used in a GST–pVHL pull-down assay. IB, immunoblotting.

pVHL disrupts the RACK1–IGF-IR complex

As pVHL and IGF-IR associate with pVHL in a reciprocal manner, we proposed that pVHL competes with IGF-IR for RACK1 binding. To test this hypothesis, 293T cells were transfected with expression plasmids for HA-IGF-IR (1 μg) and with increasing amounts of HA-VHL (0–1 μg). RACK1-associated pVHL and IGF-IR were determined by IP with anti-RACK1, followed by western blot with anti-HA. Expression of pVHL had no effect on the expression level of RACK1 and IGF-IR. However, binding of IGF-IR to RACK1 was dramatically reduced, concomitant with an increase of RACK1–pVHL complex formation (Figure 5c). To exclude the possibility that pVHL competes with RACK1 for IGF-IR binding, we examined interaction of pVHL with IGF-IR in a GST–pVHL pull-down assay. Results showed that pVHL did not bind to IGF-IR (Figure 5d). In control experiments, RACK1 was shown to bind to GST–pVHL as described previously. These data strongly suggest that pVHL can compete with IGF-IR and dissociate IGF-IR from RACK1 in vivo.

pVHL-deficient RCC cells exhibit greater PI3K/AKT activation upon IGF-I treatment

The PI3K/AKT cascade is a major downstream signaling pathway upon IGF-IR tyrosine kinase activation. It has been demonstrated that phosphorylated IRS-2 interacts with PI3K and mediate its activation (Shaw, 2001; Rajala et al., 2004). Thus, we decided to investigate whether increased IGF-IR kinase activation and IRS-2 phosphorylation observed in pVHL-defective RCC cells was associated with changes in PI3K/Akt activation. Western blot analysis with phospho-473 specific Akt antibody revealed increased Akt activity upon IGF-I treatment in both pVHL-positive and pVHL-deficient RCC cells. There was also greatly enhanced IGF-I-induced Akt activation in pVHL-deficient RCC cells, which was reduced when cells were transiently transfected with RACK1 siRNA (Figure 6). Together, these results indicate that there are alterations of the IGF-I-activated PI3K/Akt signal pathway in pVHL-deficient RCC cells and it is correlated with changes in the IGF-IR–RACK1 interaction.

Figure 6

RACK1 siRNA inhibits the IGF-I-induced Akt phosphorylation in pVHL-deficient RCC cells. RCC cells with different pVHL status were transiently transfected with or without RACK1 siRNA. Cells were then serum-starved followed by stimulation with IGF-I for indicated times. The cell lysates were then harvested and resolved by SDS–polyacrylamide gel electrophoresis and analyzed by western blotting with anti-phospho-Akt (P-Akt). IB, immunoblotting.

pVHL-deficient RCC cells exhibit increased IGF-I-induced MMP-2 expression and activity

It has been suggested that increased IGF-IR signaling is associated with activation of MMP-2 and increased cellular invasiveness via the PI3K/Akt pathway. Therefore, we collected the culture supernatant from RCC cells with different pVHL statuses after IGF-I stimulation and measured the MMP-2 activity using MMP-2 activity assay plates. MMP-2 protein expression levels in these cells were determined by western blot analysis. After IGF-I treatment, pVHL-deficient RCC cells demonstrated much more increased MMP-2 expression and activation than the pVHL-positive RCC cells (Figure 7a).

Figure 7

Loss of pVHL is associated with increased IGF-I-induced MMP-2 expression and activity. (a) RCC cells with different pVHL status were exposed to IGF-I for indicted intervals. ELISA assays were performed to measure the MMP-2 activity in the culture supernatants and western blot analysis was used for MMP-2 expression in these cells. Note that loss of pVHL is associated with increased IGF-I-induced MMP-2 expression (upper panel) and activity (lower panel). (b) Both RACK1 siRNA and specific PI3K/Akt inhibitor LY294002 suppressed the IGF-I-induced MMP-2 activation in pVHL-deficient RCC cells. pVHL-deficient RCC cells were pretreated with RACK1 siRNA, LY294002 or a mock control. Cells were then serum-starved followed by stimulation with IGF-I for indicated times. MMP-2 expression (upper panel) and activity (lower panel) were determined as described in (a). IB, immunoblotting.

To investigate whether the IGF-I–RACK1 interaction and the PI3k/Akt pathway were involved in the upregulation of MMP-2 activity and expression, we also applied the RACK1 siRNA and PI3K-specific pharmacological inhibitor LY294002 (20 μM) to pVHL-defective RCC cells for different intervals before determining the IGF-I-induced MMP-2 activity and expression. Our results showed that both RACK1 siRNA and LY294002 suppressed the IGF-I-induced MMP-2 activity in pVHL-deficient RCC cells by at least 70%. They also dramatically decreased the MMP-2 expression in these cells (Figure 7b).

pVHL-deficient RCC cells demonstrate enhanced IGFI-stimulated cellular invasiveness

IGF-IR has increasingly been recognized as an important regulator of cancer cell invasion (Dunn et al., 1998; Brodt et al., 2001; Loughran et al., 2005). pVHL-deficient RCC cells have been reported to be highly invasive upon stimulation with some growth factors(Koochekpour et al., 1999). To determine the effect of loss of pVHL in RCC cells on IGF-I-induced cellular invasiveness, the matrigel chamber invasion assay was performed. Our results demonstrated that pVHL-deficient RCC cells possessed increased cellular invasiveness toward IGF-I by approximately fivefold compared with pVHL-positive RCC cells (Figure 8). We then confirmed that RACK1 was an important mediator for this increase in IGF-I-stimulated invasiveness by transient transfection of the pVHL-defective RCC cells with RACK1 siRNA or control scrambled siRNA, before performing the cellular invasive assay. Incubation with RACK1 siRNA, but not the scrambled siRNA, reduced IGF-I-induced pVHL-deficient RCC cellular invasiveness by >40%. All these data suggest loss of pVHL promotes an IGF-I-induced invasive potential in RCC cells via the upregulation of the IGF-I signaling pathway and it is, at least, partially RACK1-dependent.

Figure 8

pVHL blocks IGF-I-induced RCC cell invasion partially through the regulation of RACK1. pVHL-positive and pVHL-deficient RCC cells were exposed to IGF-I either in the presence of RACK1 siRNA or the non-specific scrambled control siRNA. Matrigel assays were performed after 48 h stimulation. (a) Representative fields of Matrigel assays. (b) Quantitative analysis of the Matrigel assay results.


In this report, we show that the RACK1 is a pVHL-interacting protein and a key regulator of the elevated IGF-I-induced cellular invasiveness seen in pVHL-defective RCC cells. Numerous studies have shown that overactivation of IGF-IR receptor tyrosine kinase is associated with increased cellular invasiveness and metastasis of human tumors (Long et al., 1998), while inhibition of IGF-IR signaling inhibits tumor growth and enhances chemotherapy and radiation responses (Min et al., 2005; Wang et al., 2005b). RACK1 was identified as an IGF-IR interacting protein and was found to be upregulated in several metastatic cancers (Berns et al., 2000). It has also been demonstrated that RACK1 regulates IGF-IR signaling and enhances tumor cell spreading and migration (Kiely et al., 2005). Thus, the finding that RACK1 forms a complex with pVHL prompts us to investigate the effect of the association between RACK1 and pVHL on IGF-I-stimulated RCC cell invasiveness. Our studies show that, in addition to its roles as an E3-ubiquitin ligase component regulating hypoxia-inducible genes expression, pVHL also involves in the modulation of the IGF-IR signal pathways via the interaction with RACK1. Importantly, we find that loss of pVHL leads to increased IGF-IR kinase activity and cellular invasiveness, which can be reversed by knockdown of RACK1 expression. These findings are consistent with previous reports that VHL−/− RCC cells are highly invasive upon growth factor treatment (Koochekpour et al., 1999).

RACK1 contains seven WD40 repeat domains. The fact that multiple proteins interact with different WD40 domains on RACK1 suggests RACK1 is a signal mediator with several docking domains (McCahill et al., 2002). A recent report has shown that RACK1 binds to IGF-IR through WD1–4 (Zhang et al., 2006). Our data demonstrate that the domains necessary for its association with pVHL are the WD6–7 domains. Interestingly, we found that in pVHL-deficient cells, there was increased RACK1 IGF-IR association upon IGF-I treatment, which can be disrupted by enhanced pVHL expression level. Moreover, pVHL-deficient cells demonstrate elevated IGF-IR kinase activity that can be reversed by RACK1 siRNA. We also found pVHL competes with IGF-IR for RACK1 interaction. These observations suggest that pVHL functions as a negative regulator of IGF-I signaling by interacting with RACK1 and preventing it from binding to IGF-IR. These observations also imply that RACK1 may have a role in activating IGF-IR signal pathways by a mechanism that requires its dissociation from pVHL.

It has been demonstrated that RACK1 binds to elongin C and recruits the elongin C/B ubiquitin ligase complex to promote ubiquitination of HIF-1α through an O2 and pVHL-independent mechanism (Liu et al., 2007). The ability of RACK1 acting as a scaffold to interact with a wide variety of signaling proteins probably reflects its ‘crossroad-traffic-control’ role to integrate and direct communication from different signaling cascades. Based on previously published data and data presented here, It is conceivable that RACK1 may regulate different signaling pathways via creating a competition between signaling proteins for interaction with RACK1, which regulates the balance between antagonistic or different signaling pathways. Alternatively, individual proteins may bind RACK1 under discrete conditions, such as normoxia or hypoxia, to control different downstream pathways.

Cell invasion involves the activation and secretion of proteolytic enzymes such as MMPs, which lead to reorganization of the extracellular matrix components. Studies have indicated that IGF-I induces elevated expression and activation of MMPs, including MMP-2, in tumor cells (Yoon and Hurta, 2001; Stawowy et al., 2004). Meanwhile, MMPs have been shown to be the key enzymes for tumor progression in RCC and increased MMP-2 expression levels are observed from advanced carcinoma samples (Kugler et al., 1998; Kugler 1999). In our study, stronger MMP-2 expression and activation in response to IGF-I stimulation was seen in pVHL-deficient RCC cells. Elevated level of MMP-2 has also been reported in pVHL-deficient RCC cells upon hepatocyte growth factor stimulation (Koochekpour et al., 1999). Thus, overactivation of MMP-2, due to enhanced IGF-IR or other growth factor signals induced by loss of pVHL, may therefore contribute to the increased cellular invasiveness in these cells. Interestingly, in pVHL-deficient RCC cells, invasiveness toward 20 ng/ml hepatocyte growth factor could not be significantly suppressed by RACK1 siRNA (data not shown), indicating that the role RACK1 in regulating cellular invasiveness is likely extracellular signal specific.

The activation of IGF-IR can trigger multiple signaling pathways including PI3K/Akt, which is implicated in induced expression of MMPs, cell migration and tumor metastasis (Brader and Eccles, 2004). Our studies show that upon IGF-I treatment, while the PI3K/Akt pathway is activated in both the pVHL-deficient and pVHL-positive RCC cells, pVHL-deficient cells manifest greater activation that can be reversed by RACK1 siRNA. We also observed that elevated MMP-2 expression and activation seen from these pVHL-deficient cells could be reversed by the application of the specific PI3K inhibitor LY294002. These observations indicate that the pVHL–RACK1 interaction has an important role in IGF-I-induced PI3K activation, which is involved in augmenting MMP-2 production and activation. Whereas it seems plausible that the pVHL–RACK1 interaction regulates the IGF-I-induced PI3K activation via the modulation of IGF-IR tyrosine kinase activity, other potential RACK1-mediated ways of regulation may still exist. For example, several studies show Src kinase, one of the RACK1-interacting proteins (Chang et al., 2002), is a downstream target of IGF-IR signaling and is involved in tumor cell invasion (Sekharam et al., 2003). Meanwhile, Src has been shown to directly interact with and activate Akt. Therefore, the pVHL–RACK1 interaction could potentially modulate the IGF-I-induced Akt signaling pathway via the interplay between Src and Akt. Further studies are required to define a role for Src in the IGF-I-Akt signaling pathway in these cells. Additionally, the fact that our results showed only partial block of the RCC cell invasiveness with knockdown of RACK1 expression suggests that other pVHL-related signal pathways might be involved in IGF-I-induced RCC cell invasiveness.

In summary, we have demonstrated that pVHL interacts with RACK1, which leads to disruption of the association between RACK1 and IGF-IR, and suppression of RACK1-mediated IGF-I-induced RCC cell invasiveness. This increased invasiveness is associated with enhanced IGF-IR kinase activity and elevated MMP-2 expression and activation. This activation pathway is RACK1 dependent. Our data indicate that RACK1 serves as a direct mediator between loss of pVHL function and enhanced IGF-IR signaling pathway in RCC.

Materials and methods

Yeast two-hybrid screens

The GAL4 Two-Hybrid Systems and pretransformed human kidney cDNA library (BD Bioscience Clontech, Palo Alto, CA, USA) were used to identify potential protein interactions. The full-length human VHL cDNA sequence was cloned in frame into the GAL4DNA-BD vector to generate the bait construct. The bait plasmid was transformed into the AH109 yeast strain and its protein expression was confirmed by western blot. Positive mating zygotes with β-galactosidase activity were selected on synthetic –Trp/-Leu/-His/-Ade dropout (QDO) agar plates. Plasmids from positive colonies were isolated and retransformed into the yeast strain Y190 with either mock vector or GAL4DNA-BD-VHL to confirm that growth on QDO and β-gal activity was VHL-dependent. The cDNA inserts from positive clones were subjected to DNA sequencing with a dye terminator cycle sequencing kit (University of Rochester Research Core Facility, Rochester, NY, USA).

Cells and cells culture

All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) plus penicillin/streptomycin. Human kidney 293T cells and human renal cancer cell lines 786-O, Caki1 and ACHN were obtained from American Type Culture Collection (Manassas, VA, USA). 786-O is a pVHL-defective sporadic RCC cell line. Its sublines 786-O-pVHL+ (WT8) and 786-O-pVHL- (pRC3) were created by stable transfection of wild-type HA-VHL or empty vector, respectively (obtained from Dr William Kaelin Jr, Dana-Farber Cancer Institute, Boston, MA, USA). Two stable RCC4 renal cancer cell lines expressing HA-pVHL or the mock vector, RCC4-pVHL+ and RCC4-pVHL-, were provided by Dr Maxwell (Imperial College, London, UK).

Stimulation of cell

RCC cells or transient transfected 293T cells grown to near confluence were incubated in serum-free DMEM for 24 h. The cells were then incubated in serum-free DMEM supplemented with 20 ng of IGF-I (Sigma, St Louis, MO, USA) per ml for intervals as indicated in the figure legends. Cells were then immediately placed on ice and protein extracts were prepared for IP and western blot analysis. The PI3K-specific inhibitor, LY294002, was purchased from Sigma.

Plasmid construction and transfection

Cells were transiently transfected with various combinations of plasmids using lipofectamine2000 reagent (Life Technologies, Inc., Rockville, MD, USA; Wang et al., 2005a). Primer pairs 5′-IndexTermATCAAGCTTATGACTGAGCAGATGACCC-3′ (sense) and 5′-IndexTermGCGTCGACCTAGCGTGTGCCAAT-3 (antisense), 5′-IndexTermTATCCCGGGTATGACTGAGCAGATGACCC-3′ (sense) and 5′-IndexTermATCCCGGGCTAGCGTGTGC-3′ (antisense) and human kidney cDNA library were used to generate full-length RACK1 PCR fragments. The fragments were then cloned into the mammalian expression vector pCMV-Tag 2 (Strategene, La Jolla, CA, USA) and pKH3 to generate FLAG-RACK1 and HA-RACK1 fusion protein constructs. Expression plasmids for wild-type pVHL were described previously (Li et al., 2002). Plasmids for GFP-tagged truncated pVHL were constructed by subcloning the corresponding VHL fragments into the pEGFP vector (BD Bioscience Clontech, Palo Alto, CA, USA) after PCR amplifications. All constructs were verified by DNA sequencing.

Preparation of cellular protein extracts and IP

Cells were placed on ice and washed twice with cold phosphate-buffered saline, then lysed in RIPA buffer (1 × phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with complete protease inhibitors (Roche Applied Science, Indianapolis, IN, USA). Total protein concentration of each sample was measured using the Bradford assay kit (Bio-Rad, Hercules, CA, USA). For co-IP, 500 μg of whole cell extracts were precleared for 30 min with 10 μl protein A/G PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA), together with 0.2 μg of the control IgG. The lysates were recovered by centrifugation and incubated with the indicated primary antibodies agarose conjugates for 4 h (for in vitro kinase assay) or overnight at 4 °C with gentle rocking. Immunoprecipitates were washed three times in fresh cold lysis buffer and then either used for in vitro kinase assay or removed from the beads by 5 min boiling in SDS sample buffer (360 mM Tris, pH 6.6; 600 mM dithiothreitol; 60% glycerol; 0.6% bromophenol blue; 10% SDS) for SDS–polyacrylamide gel electrophoresis and western blot analysis.

Western blot analysis

Total cellular lysates or immunoprecipitates (20–60 μg) were electrophoretically resolved on denaturing SDS–polyacrylamide gels, transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in TBS-T containing 5% skim milk and probed with following antibodies: anti-HA HRP, anti-GST and anti-GFP HRP (Santa Cruz Biotechnology), anti-RACK1 (BD Transduction Laboratories, Heidelberg, Germany), anti-FLAG and anti-β-actin (Sigma, St Louis, MO, USA), anti-Akt and anti-phospho-Akt (Ser-473) (Cell Signaling, Beverly, MA, USA), mouse anti-human MMP-2 antibody (EMD Biosciences, San Diego, CA, USA). Other antibodies used in our experiments are anti-IGF-IR antibody (Chemicon, Temecula, CA, USA), mouse monoclonal anti-phosphotyrosine antibody 4G10 and anti-IRS-2 antibody (Upstate Biotechnology, Lake Placid, NY, USA). The signals were visualized by the ECL chemiluminescence detection system (Amersham Bioscience Corp., Piscataway, NJ, USA) following the manufacture's instruction.

Hypoxia experiment

Cells in DMEM medium supplied with 10% fetal bovine serum were exposed to constant flow of 1% O2, 5% CO2 and 94% N2 in a humidified hypoxia incubator (ThermoForma, Forma Scientific Inc., Waltham, MA, USA) at 37 °C. To analyze protein degradation kinetics, the cells were maintained in hypoxia for 4 h before released into normoxia for 10 min with the addition of 20 μg/ml cycloheximide. Then the cells were immediately lysed and the protein samples were harvested for western blot analysis.

RNA interference

siGENOME SMARTpool targeted to VHL was used (Dharmacon, Austin, TX, USA). The nonspecific siCONTROL Non-Targeting siRNA Pool (Dharmacon, Inc., Lafayette, CO, USA) was used as negative control. HEK293A (VHL+/+) cells grown on six-well tissue culture plates were transfected with scrambled and VHL siRNA at a final concentration of 200 nM. Four human RACK1 (accession no. NM_006098) siRNAs were designed according to siRNA design guideline and SiRNA Target Finder (Ambion, Inc., Austin, TX, USA). These targeted sequences are 5′-43IndexTermAAGCCATCCAGTGCCATCC61-3′, 5′-143IndexTermAACGGCTGGGTAACCCAGATC163-3′, 5′-233IndexTermAAACTGACCAGGGATGAG250-3′ and 5′-506IndexTermAAGCTATGGAATACCCTGGGT526-3′. The mixture of the siRNAs led to significant silence of the RACK1 expression and was used in all related experiments.

Assay of IGF-I receptor kinase activity

The IGF-IR kinase activity was measured using Poly (Glu-Tyr) (Sigma) as the substrate. Cells were serum starved for 6 h and then treated with IGF-I for 0, 15 or 30 min. Cell lysates were immunoprecipitated with anti-IGF-IR antibody as described above. The immune precipitates were washed three times in the kinase buffer (50 mM Hepes pH 7.6, 150 mM NaCl, 0.05% bovine serum albumin, 0.1% Triton X-100, 10 mM MgCl2 and 5 mM MnCl2), which did not reduce the amount of bound IGF-IR as checked by western blot analysis. A mock immunoprecipitate containing no cell lysate was served to blank the assay. The beads were then resuspended in 20 μl of the kinase reaction mixture (50 mM HEPES pH7.6, 150 mM NaCl, 0.05% bovine serum albumin, 0.1% Triton X-100, 10 mM MgCl2, 5 mM MnCl2, 1 mg/ml Poly (Glu-Tyr) and 5 μCi of carrier-free [γ-32P] ATP). Following 20 min incubation at 25 °C with constant mixing, the reaction was terminated and 10 μl of the reaction mixture was spotted onto Whatman P81 phosphocellulose strips. The strips were washed three times in 10% trichloroacetic acid containing 1% H3PO4 and the radioactivity was quantitated using a Beckman scintillation analyzer (Beckman Instruments, Fullerton, CA, USA).

MMP-2 activity assay

MMP-2 activity in cell culture supernatant was quantified after 6 or 12 h culture in fresh serum-free DMEM containing IGF-I or phosphate-buffered saline mock vehicle, using MMP-2 Biotrack Activity Assay System (Amersham Bioscience Corp.) in accordance with the manufacture's instruction. Plates were read at 405 nm using Berthold luminometer (EG&G Wallac, Gaithersburg, MD, USA). Three samples were counted for each determinant and experiments were performed triplicate.

Immunofluorescence staining

Human kidney tissue paraffin slides were de-waxed and then incubated with the primary antibody overnight at 4 degree and, after three washes, were incubated with a mixture of secondary antibodies (FITC-conjugated goat anti-mouse IgG and rhodamine-conjugated goat anti-rabbit IgG; Wang et al., 2007). After washing, the slides were mounted in Vectashield and analysed under a Zeiss Auxiphot epifluorescence microscope.

In vitro cellular invasive assay

Cell invasion assays were performed using the BioCoat Growth Factor Reduced Matrigel invasion chamber with 8 μM pore size (BD Biosciences, Bedford, MA, USA). Briefly, the upper inserts were rehydrated with warm culture medium for 2 h. Same amount (2.5 × 104) of cells in serum-free medium containing 0.1% bovine serum albumin were added to each chamber and allowed to migrate toward the underside of the membrane for 48 h with or without IGF-I in the lower chamber as the chemo attractant. After cells on the upper surface of the membrane were removed by wiping with a cotton swab, migrated cells were fixed and stained in Diff-Quick solution (BD Biosciences). The relative invasion was calculated as the number of invading pVHL-defective cells that had passed to the lower surface of the membranes was assigned a value of 1.0 in each experiment. A total of four random fields per membrane were counted for each assay. Every determination represents the mean of three separate experiments. The effect of RACK1 siRNA was determined by transient transfection of the RACK1 siRNAs or the non-specific scrambled siRNA into the cells before performing the invasion assay.

Accession codes




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We thank Dr Patrick H Maxwell and Dr William G Kaelin Jr for their generous gifts of the RCC stable cell lines. We also thank Christopher R Silvers for his technical support. Funding: This work was supported in part by the James P Wilmot Cancer Center grant to X He.

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Correspondence to G Wu.

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  • VHL
  • RACK1
  • IGF-I
  • renal cell carcinoma

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