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. Author manuscript; available in PMC: 2011 Sep 10.
Published in final edited form as: Mol Cell. 2010 Sep 10;39(5):797–808. doi: 10.1016/j.molcel.2010.08.016

Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction

Daming Gao 1, Lixin Wan 1, Hiroyuki Inuzuka 1, Anders H Berg 1, Alan Tseng 1, Bo Zhai 2, Shavali Shaik 1, Eric Bennett 3, Adriana E Tron 4, Jessica A Gasser 1, Alan Lau 1, Steven Gygi 2, J Wade Harper 3, James A DeCaprio 4, Alex Toker 1, Wenyi Wei 1,5
PMCID: PMC2939073  NIHMSID: NIHMS230171  PMID: 20832730

Summary

The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s), remains largely unknown. Here we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers.

Introduction

The mammalian target of Rapamycin (mTOR) plays a critical role in regulation of cellular homeostasis, cell growth and survival pathways by acting as a sensor for upstream inputs from multiple growth-promoting signals which are then transduced to downstream effectors (Guertin and Sabatini, 2007; Reiling and Sabatini, 2006). In order to fulfill this complex regulatory function, the mTOR kinase assembles into at least two distinct complexes termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Guertin and Sabatini, 2007; Reiling and Sabatini, 2006). These two multi-component subcomplexes differ both structurally and functionally and signal to distinct downstream substrates. mTORC1 is composed of mTOR, Raptor, PRAS40 (proline-rich Akt substrate of 40 kilodaltons) and mLST8/GβL (G protein β-subunit-like protein). The best characterized mTORC1 kinase substrates include S6K (p70 S6 ribosomal kinase) and 4E-BP1 (phosphorylated 4E-binding protein). The mTORC2 complex includes mTOR, Rictor, mLST8/GβL, PROTOR (protein observed with Rictor-1)/PRR5 and Sin1 (Jacinto et al., 2006; Shiota et al., 2006). mTORC2 phosphorylates the hydrophobic motif of Akt at Ser473 (Sarbassov et al., 2005) and SGK1 at Ser422 (Garcia-Martinez and Alessi, 2008), leading to full kinase activation. Since aberrant activation of Akt is a hallmark of many types of cancers (Manning and Cantley, 2007), hyperactivation of mTORC2 activity has been implicated in cancer progression (Guertin and Sabatini, 2007).

The activity of the mTORC1 complex is highly regulated in cells exposed to growth factors and nutrients. In response to mitogens, activation of PI 3-K (phosphoinositide 3-kinase) leads to phosphorylation of the TSC2 (tuberous sclerosis 2) and PRAS40 proteins by Akt, culminating in activation of mTORC1 (Manning and Cantley, 2007). The activity of mTORC1 can also be stimulated by the Rag GTPase in response to nutrient stimulation (Sancak et al., 2008). Additionally, phosphorylation of Raptor by AMPK (5′ AMP-activated protein kinase) in response to a low energy state provides a negative regulatory mechanism to repress mTORC1 activity (Gwinn et al., 2008). Although mTORC2 is a key upstream kinase complex that functions to control Akt phosphorylation and downstream signaling, relatively little is known regarding the regulation of mTORC2. Recent studies indicate that the mTOR complexes might be multi-functional and contain activities other than protein kinases. For example, Raptor has been shown to form a complex with the Cullin-4 E3 ligase and this complex might be critical for mTOR kinase activity (Ghosh et al., 2008). Rictor has also been shown to associate with Cullin-4, although unlike Raptor it is not a WD40-repeat-containing protein (Ghosh et al., 2008). However, additional function(s) for Rictor and mTORC2 remain largely unknown.

The serum and glucocorticoid-inducible kinase (SGK) belongs to the AGC (protein kinase A, G and C) family of kinases, and its activity is stimulated by growth factors (Lang et al., 2006). There are three closely related family members designated SGK1, SGK2 and SGK3 (Loffing et al., 2006). One of the best characterized SGK1 downstream targets is Foxo3a, which is involved in the regulation of apoptosis (Brunet et al., 2001). SGK1 has also been indicated in the regulation of Na+ retention through phosphorylation of Nedd4-2 to impair its ability to degrade the epithelial Na+ channel (ENaC) (Debonneville et al., 2001; Ichimura et al., 2005). SGK isoforms share approximately 80% similarity in the kinase domains with other AGC family kinases including Akt and S6K. In vitro SGK recognizes the same phosphorylation consensus motif (RXRXXS/T, where X represents any amino acid) as Akt and S6K (McCormick et al., 2004). However, unlike Akt and S6K whose expression is relatively stable, SGK1 is a short-lived protein whose stability is controlled by the ubiquitin-proteasome pathway (Loffing et al., 2006). Both the Nedd4-2 (Brickley et al., 2002; Zhou and Snyder, 2005) and CHIP (C-terminal Hsc70-interacting protein) E3 ligases have been shown to ubiquitinate SGK1 (Belova et al., 2006). The first 60 amino acids of SGK1 are critical for Nedd4-2 mediated destruction of SGK1 (Bogusz et al., 2006; Brickley et al., 2002). In addition to growth factor stimulation, cellular stresses including osmotic stress, heat shock, oxidative stress and ultraviolet irradiation induce SGK1 expression by transcriptional mechanisms and thus influence cell survival, proliferation and differentiation (Lang and Cohen, 2001; Loffing et al., 2006). However, it remains unclear how SGK1 destruction by Nedd4-2 and CHIP are regulated by these cellular stresses and whether other unidentified E3 ligase(s) play a critical role in governing SGK1 destruction in response to these signaling events. Finally, both Akt and SGK are frequently amplified and/or overexpressed in cancers (Sahoo et al., 2005), although the underlying molecular mechanisms are unknown.

Here we evaluate the mechanism by which Rictor controls SGK1 stability. We found that by specific association with Cullin-1 and Rbx1, Rictor forms a functional E3 ubiquitin ligase complex that promotes the ubiquitination of SGK1, but not Akt1 or S6K1. We also show that the AGC kinases phosphorylate Rictor at T1135 to disrupt the Rictor/Cullin-1 complex and impair its E3 ligase activity and subsequent SGK1 ubiquitination. Our findings demonstrate a kinase-independent function for the Rictor protein and provide a mechanistic explanation for the observed elevation of SGK1 expression in various human tumors.

Results

SGK1 Protein Expression is Regulated by Rictor

SGK1 is an unstable protein and previous studies have shown that in response to serum and growth factors, activation of PI 3-K leads to the induction of SGK1 (Park et al., 1999). Induced SGK1 expression occurs partially through the increased transcriptional levels of SGK1 mRNA, and partially through other layers of post-translational regulation (Loffing et al., 2006). However, the exact molecular mechanism(s) remain elusive. Recent studies have shown that the Rictor-containing mTORC2 complex phosphorylates SGK1 at S422 in the hydrophobic motif to fully activate SGK1 kinase activity (Garcia-Martinez and Alessi, 2008). We therefore first investigated if Rictor signaling can also influence SGK1 expression. We found that Rictor−/− MEFs have elevated SGK1 expression levels under serum-deprived conditions (Figure 1A). In agreement with previous studies (Webster et al., 1993), re-addition of serum led to a significant induction of SGK1 protein in both wild type and Rictor −/− MEFs. Elevated SGK1 expression in Rictor −/− MEFs has been reported (Huang et al., 2009), although the underlying molecular mechanism has not been characterized. Loss of Rictor is the primary mechanism for the observed elevation of SGK1 expression since re-introduction of wild-type Rictor into Rictor−/− MEFs dramatically reduced SGK1 expression (Figure 1B). Consistent with this, depletion of Rictor, but not Raptor, in HeLa cells also led to an accumulation of SGK1 protein, primarily in the early G1 phase of the cell cycle (Figure 1C). However, Akt1 and S6K1 expression was not affected by depletion of endogenous Rictor in HeLa cells (Figure 1C). Depletion of Rictor does not significantly affect SGK1 mRNA levels (Figure S1A–C), indicating that post-translational modification(s) might contribute to the regulation of SGK1 by Rictor. In support of this notion, depletion of Rictor led to a significant increase in SGK1 half-life (Figure S1H). Furthermore, depletion of other mTORC2 complex components mTOR or Sin1 resulted in a decrease, rather than accumulation of SGK1, indicating that Rictor might regulate SGK1 abundance independent of its mTORC2 kinase activity (Figure S1D). Moreover, inactivation of the P-I3K pathway by both LY and Wortmannin treatment leads to a significant decrease in SGK1 expression and this process was blocked by MG132 treatment (Figure S1E–F), supporting an involvement of the 26S-proteasome pathway. More importantly, LY-induced SGK1 degradation is partially blocked after depletion of the endogenous Rictor (Figure 1D), further supporting a potential physiological role for Rictor in SGK1 stability control.

Figure 1. SGK1 expression is regulated by the Rictor pathway.

Figure 1

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A. Whole cell lysates were isolated from wild type or Rictor −/− mouse embryonic fibroblasts (MEFs) in serum starvation conditions for 24 hours. In another experimental condition, 10% FBS was added to the serum-starved cells for 1.5 hours before harvesting. Equal amounts of whole cell lysates were immunoblotted with the indicated antibodies.

B. Immunoblot analysis of wild type or Rictor−/− MEFs transfected with the Myc-Rictor plasmid (with empty vector as a negative control) together with pBabe-Puro retroviral empty vector constructs. Twenty-four hours post-transfection, the cells were treated with 1 μg/ml puromycin for 48–72 hours to kill the non-transfected cells prior to collecting the whole cell lysates for immunoblots.

C. HeLa cells were infected with the indicated lentiviral shRNA constructs and selected with 1μg/ml puromycin to eliminate the non-infected cells. The resulting HeLa cell lines were arrested in the M phase by incubation with nocodazole for 18 hours and then released into the G1 phase. At the indicated time points, cell lysates were collected for immunoblot analysis.

D. HeLa cells were infected with lentiviral shRictor construct (with shGFP as a negative control) and selected with 1 μg/ml puromycin to eliminate the non-infected cells. The resulting cell lines were treated with 20 μM LY294002, and at the indicated time points, whole cell lysates (WCL) were collected for immunoblot analysis.

(see also Figure S1)

To better understand how Rictor controls SGK1 expression, we examined the difference between wild-type and Rictor−/− MEFS in their endogenous signaling pathways responding to growth factor addition (insulin in Figure S1I and IGF in Figure S1J). We found that Rictor−/− MEFs are defective in activating Akt, as evidenced by the lack of induction of the pSer473-Akt signal (Guertin et al., 2006; Shiota et al., 2006). Interestingly, SGK1 expression in wild-type MEFs peaks 0.5–1 hour post-growth factor addition, and then fades away at later time points (2- 4 hour). In contrast, there is elevated SGK1 expression in Rictor−/− cells and no significant decrease of SGK1 at the late time-points. This data suggests a possible role for Rictor in promoting the destruction of SGK1 at late time-points following growth factor addition (Lang and Cohen, 2001; Webster et al., 1993).

Rictor Promotes SGK1 Ubiquitination in a Cullin-1-dependent Manner

In support of the finding that Rictor regulates SGK1 stability, we detected an interaction between Rictor and SGK1 (Figure 2A). Ubiquitin immunoblotting reveals that Rictor can promote SGK1 ubiquitination (Figure S2A). To exclude the possible contribution of other known SGK1 E3 ligases, including Nedd4-2 and CHIP, we used a deletion mutant of SGK1 that lacks the amino-terminal 60 amino acids (Δ60-SGK1), which cannot be efficiently ubiquitinated by Nedd4-2 and CHIP (Brickley et al., 2002; Zhou and Snyder, 2005). We used a cell-based ubiquitination assay to address whether Rictor and other mTOR components could promote SGK1 ubiquitination. Surprisingly, E3 ligase activity towards SGK1 is unique to Rictor as neither Raptor nor mTOR promotes SGK1 ubiquitination (Figure 2B). SGK1 was recently identified to be a specific downstream substrate of mTORC2 (Rictor), but not mTORC1 (Raptor) (Garcia-Martinez and Alessi, 2008). Therefore, we reasoned that SGK1 may specifically interact with Rictor, but not Raptor, which could explain why Raptor does not promote SGK1 ubiquitination, despite the fact that Raptor was recently shown to exist in a complex with Cullin 4 (Ghosh et al., 2008). However, as shown in Figure S2B, Raptor does not promote the ubiquitination of either S6K1 or Akt1, consistent with the widely accepted notion that unlike SGK, both Akt and S6K are relatively stable proteins. Furthermore, short-term or long-time treatment with Rapamycin does not affect the ability of Rictor to promote SGK1 ubiquitination, even though long-term Rapamycin treatment led to a significant decrease of SGK1 Ser422 phosphorylation by mTORC2, arguing that Rictor-mediated SGK1 ubiquitination might be mTORC kinase activity-independent (Figure 2C).

Figure 2. Rictor promotes SGK1 ubiquitination in a Cullin-1-dependent manner.

Figure 2

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A. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with Flag-Δ60-SGK1 and Myc-Rictor constructs. Thirty hours post-transfection, cells were treated with 10 μM MG132 for 10 hours to block the proteasome pathway before harvesting.

B. Immunoblot analysis of whole cell lysates (WCL) and anti-HA immunoprecipitates derived from 293T cells transfected with the indicated plasmids. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight before harvesting.

C. Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-HA immunoprecipitates of 293T cells transfected with the indicated plasmids. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight, or 100 nM Rapamycin for the indicated period of time before harvesting.

D. Expression of a dominant negative form of Cullin-1 blocks the ability of Rictor to promote SGK1 ubiquitination. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Δ60-SGK1, Myc-Rictor and His-Ub in the presence of various dominant negative forms of Cullin family members. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight before harvesting. The whole cell lysates were collected in EDTA-free lysis buffer and the His-pull down was carried out in the presence of 8 M Urea to disrupt possible protein interactions.

E. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated Myc-Cullin constructs. Thirty hours post-transfection, cells were treated with 10 μM MG132 for 10 hours to block the proteasome pathway before harvesting.

(see also Figure S2)

Since Rictor itself does not contain the ring-finger, PHD or HECT domain that possesses intrinsic E3 ligase activity, it is possible that Rictor associates with other co-factor(s) to form an E3 ligase complex. The Cullin-Ring complex comprises the largest family of E3 ubiquitin ligases (Petroski and Deshaies, 2005). Thus, we first determined which Cullin family member might contribute to SGK1 ubiquitination. We found that overexpression of a dominant negative Cullin-1 allele (but not DN Cullin 2, 3 or 4) specifically impairs the ability of Rictor to promote SGK1 ubiquitination (Figure 2D). In keeping with this finding, we showed that Rictor specifically interacts with Cullin-1, but not other Cullin family members Cullin-2, 3 and 4 (Figure 2E and S2C–D). These data are indicative of a role for Cullin-1 in SGK1 ubiquitination.

Rictor forms a complex with Cullin-1, Rbx1 to promote SGK1 destruction

In support of a possible physiological role for both Rictor and Cullin-1 in regulating SGK1 stability, we detected the endogenous interaction between Rictor and Cullin-1 (Figure 3A–B). Furthermore, the endogenous interaction between Rictor and Cullin-1 could be detected in both CHAPS buffer that preserves the mTORC2 complex (Figure 3A and S3A), and NP-40 containing EBC buffer (Figure 3B), a condition that has been shown to disrupt the mTORC2 complex (Hara et al., 2002; Kim et al., 2002), indicating that the intact Rictor/mTOR complex might not be required for interaction with Cullin-1. In support of this idea, we showed that Rictor, but not Sin1 or mTOR, specifically interacts with endogenous Cullin-1 (Figure 3C). Furthermore, Cullin-1 only specifically interacts with Rictor, but not Raptor, or other known mTORC2 components including mTOR, GβL and Sin1, (Figure 3D). Notably, Cullin-1 binds endogenous Rictor with similar intensity as Sin1 in both CHAPS and EBC buffer conditions (Figure S3D). Although mTOR interacts with Rictor more strongly than Cullin-1 in CHAPS buffer, mTOR/Rictor interaction is not detected in EBC buffer (Figure S3C) (Hara et al., 2002; Kim et al., 2002). These results suggest that Cullin-1 interacts with Rictor in vivo.

Figure 3. Rictor interacts with Cullin-1 and Rbx1 in vivo.

Figure 3

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A. Immunoblot (IB) analysis of 293T cell whole cell lysates (WCL) and anti-Cullin-1 immunoprecipitates (IP). Mouse IgG was used as a negative control for the immunoprecipitation procedure. WCL were collected with CHAPS buffer and IPs were washed with CHAPS buffer.

B. Immunoblot (IB) analysis of 293T cell whole cell lysates (WCL) and anti-Rictor immunoprecipitates (IP). Rabbit IgG was used as a negative control for the immunoprecipitation procedure. WCL were collected with EBC buffer and IPs were washed with NETN buffer.

C. Immunoblot (IB) analysis of 293T cell whole cell lysates (WCL) and anti-Rictor, anti-Sin1 and anti-mTOR immunoprecipitates (IP). Rabbit IgG was used as a negative control for the immunoprecipitation procedure. WCL were collected with EBC buffer and IPs were washed with NETN buffer.

D. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with the HA-Cullin-1 construct.

E. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Rbx1 construct. Thirty hours post-transfection, cells were pretreated with 10 μM MG132 for 10 hours to block the proteasome pathway before harvesting.

F. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with Myc-Rictor together with the indicated HA-Rbx1 constructs. Thirty hours post-transfection, cells were pretreated with 10 μM MG132 for 10 hours to block the proteasome pathway before harvesting.

G. Immunoblot analysis of HeLa cells transfected with the indicated siRNA oligonucleotides. The control lanes are scrambled E2F-1 siRNA and siRNA against firefly luciferase; siRNA, short interfering RNA.

see also Figure S3)

Cullin-1 is an extensively studied member of the Cullin family (Cardozo and Pagano, 2004; Harper et al., 2002; Nakayama and Nakayama, 2005). It complexes with Rbx1, Skp1 and various F-box proteins to form a multi-protein SCF (Skp1, Cullin-1, F-box protein) E3 ligase complex (Cardozo and Pagano, 2004; Schulman et al., 2000). To further understand the physiological components of the Rictor/Cullin-1 complex, we performed gel filtration chromatography. As shown in Figure S3G, we found that there might be two different pools of Rictor complex. One complex was estimated at a size over 600KD (fractions 22–26), co-migrating with activated mTOR (as evidenced by S2481 phosphorylation) and Sin1, which might represent the mTORC2 complex. The other Rictor-containing complex was detected at 300–400 KD (fractions 30–34), and co-migrates with both Cullin-1 and Rbx1, but not Sin1. Since Sin1 is required for mTORC2 complex formation (Jacinto et al., 2006; Yang et al., 2006), the lack of Sin1 precludes a possible existence of the mTORC2 complex in these fractions. In contrast, one Raptor complex (mTORC1 complex) is detected, estimated to be over 600KD and co-migrating with mTOR. Another detected Raptor peaks at around 180–250KD (fractions 36–40), which might represent the free Raptor monomer. Consistent with the co-immunoprecipitation data (Figure 3D), there is no detected co-migration between Raptor and Cullin-1 at fractions 29–34 with anticipated size (around 250–400KD) corresponding to a possible Raptor/Cullin-1 complex.

Consistent with the gel filtration experiment, we observed an interaction between Rictor and Rbx1 (Figure 3E and Figure S3H), which recruits the E2 enzyme to the E3 ubiquitin ligase complex. Using a series of Rbx1 mutants that are unable to interact with Cullin-1, we showed that the presence of Cullin-1 is required for Rbx1 and Rictor interaction (Figure 3F and Figure S3I). However, we found that under ectopic overexpression conditions Rictor does not interact with Skp1 (Figure S3J-K). In line with these biochemical evidences, depletion of Rictor, Cullin-1 and Rbx1, but not Skp1, leads to increased SGK1 expression (Figure 3G, S3F and S3L). Furthermore, depletion of endogenous Skp1 does not significantly affect the interaction between Rictor and Cullin-1 (Figure S3M), nor the ability of Rictor to promote SGK1 ubiquitination in vivo (Figure S3N). Altogether, these data support the hypothesis that a unique complex composed of Rictor, Cullin-1 and Rbx1 (and possibly other unknown partners) is involved in regulating SGK1 abundance. However, it requires further investigation to fully understand the role of Skp1 in this process.

We next evaluated the relationship between the mTORC2 kinase complex and the Rictor/Cullin-1 ubiquitin E3 ligase complex. In agreement with a recent study (Huang et al., 2009), both the amino- and carboxyl-termini of Rictor are required for association with mTORC2 (Figure S3O-P). In contrast, although the carboxyl-terminus of Rictor containing the T1135 site (see Figure 4A-B below) is sufficient for interaction with Cullin-1, deletion of this region did not lead to a complete loss of interaction with Cullin-1 indicating that the N-terminus of Rictor also plays a role in mediating Cullin-1 interaction (Figure S3Q). However, only the full-length Rictor, but not amino- or carboxyl-terminal truncation mutants forms a functional E3 ligase/Cullin-1 complex to promote SGK1 ubiquitination (Figure S3R). These data support a possible model whereby association of Rictor with distinct subsets of cofactors can assemble into distinct complexes to modulate a functional output, although the precise molecular mechanism that switches Rictor from a kinase mode to an ubiquitin ligase mode remains to be determined.

Figure 4. Rictor is phosphorylated in vivo at T1135.

Figure 4

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A. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated Myc-Rictor constructs together with various HA-tagged AGC family of kinases.

B. Sequence alignment of the putative Rictor T1135 phosphorylation site across different species.

C. HeLa cells were infected with the indicated lentiviral shRNA constructs and selected with 1μg/ml puromycin to eliminate the non-infected cells. Cell lysates were collected for immunoblot analysis.

D. 293T cells were transfected with the indicated Myc-tagged Rictor constructs. Thirty-six hours post-transfection, whole cell lysates were collected and the mTORC2 complex was purified by Myc-immunoprecipitation. The Myc-immunoprecipitates were incubated in vitro with the biochemically purified inactive Akt1 (from Upstate) in the presence of ATP and the kinase reaction buffer. Thirty minutes later, the reaction was stopped by the addition of the loading buffer. Akt1 phosphorylation status was examined by immunoblot analysis.

(see also Figure S4)

Rictor is Phosphorylated by AGC Kinases at T1135

Although Rictor is a key regulator of both Akt and SGK1, it remains largely unknown how its activity is regulated. In agreement with recent reports (Dibble et al., 2009; Julien et al., 2009; Treins et al., 2009), we found that S6K1 phosphorylates Rictor at T1135 (Figure 4A–B and S4A). Furthermore, ectopically expressed Rictor is phosphorylated in cells by Akt1 and SGK1 as well (Figure 4A–B). Phosphorylation of the Rictor T1135 site in vivo was further confirmed by mass spectrometry analysis (Figure S4B).

To further understand the regulatory mechanism of Rictor T1135 phosphorylation, we developed a phospho-specific antibody against this motif (Figure S4C–D). Depletion of mTOR, but not Raptor in HeLa cells, leads to a significant reduction of T1135 Rictor phosphorylation, indicating that both mTORC1 and mTORC2 activities are possibly involved (Figure 4C). In keeping with this finding, inhibition of PI 3-K with LY or Wortmannin treatment is more efficient than inhibition of Akt with a specific Akt inhibitor or inhibition of mTORC1 with Rapamycin in reducing T1135 phosphorylation (Figure S1E–G and Figure S4E). Moreover, although depletion of S6K leads to a reduction in T1135 phosphorylation, depletion of Akt1 or SGK1 delivers similar effects and depletion of any individual AGC kinase does not significantly affect endogenous Rictor T1135 phosphorylation as depletion of mTOR does (data not shown). This possibly indicates that all three AGC family kinases might function in a redundant manner towards Rictor phosphorylation at T1135. A similar mechanism has been reported for Foxo3a phosphorylation by AGC kinases (Tran et al., 2003; Vogt et al., 2005).

Phosphorylation of Rictor at T1135 disrupts the Cullin-1/Rictor interaction

Next we investigated the consequence of Rictor T1135 phosphorylation on mTORC2 complex activity. Although Rictor T1135 phosphorylation leads to recruitment of 14-3-3 (Figure S5A–C), it does not disrupt the mTORC2 complex nor does it significantly affect the ability of Rictor/mTOR to phosphorylate Akt1 in vitro (Figure 4D). These results indicate that unlike Raptor (Gwinn et al., 2008), phosphorylation of Rictor at T1135 by AGC kinase might not significantly affect its kinase activity, and likely affects distinct Rictor functions. Following this lead, we next investigated if it affects the Rictor/Cullin-1 E3 ligase complex. As shown in Figure S5E, using gel filtration chromatography, we detected co-migration of p-T1135-Rictor with an active form of mTOR (p-S2481) corresponding to the mTORC2 complex (fractions 14–20). However, p-T1135-Rictor did not co-migrate with Cullin-1 at 300–400KD (fractions 21–26). Similarly, wild-type-Rictor, but not phospho-mimetic T1135E-Rictor, co-migrated with Cullin-1 (Figure 5A and S5E), indicating that phosphorylation of Rictor at T1135 might serve to disrupt the Rictor/Cullin-1 complex. In support of this notion, Cullin-1 was found to specifically interact with non-phosphorylated Rictor species (Figure 5B–C) and mutation of Thr1135 to phospho-mimetic Glu disrupts the interaction between Rictor and Cullin-1 (Figure 5D–E and S5F).

Figure 5. Phosphorylation of Rictor at T1135 disrupts the interaction between Rictor and Cullin-1.

Figure 5

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A. HeLa cells were transiently transfected with HA-WT-Rictor and Myc-T1135E-Rictor constructs. Thirty hours post-transfection, whole cell lysates were collected in CHAPS buffer and subjected to gel filtration chromography. The relative band intensities for HA-WT-Rictor, Myc-T1135E-Rictor and p-T1135-Rictor at the indicated fractionations were quantitated. The original immunoblots were shown in Figure S5E.

B-C. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the HA-Cullin-1 construct. Anti-Rictor (B) or anti-p-T1135-Rictor (C) immunoblot analysis was performed on two identical immunoprecipitations to illustrate that Cullin-1 does not interact with T1135-phosphorylated Rictor species.

D. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with the indicated Myc-Rictor and GST-Cullin-1 constructs.

E. Autoradiography of 35S-labelled Cullin-1 bound to the indicated GST-fusion proteins.

F. HA-Cullin-1 expressing HeLa cells transiently transfected with the Flag-14-3-3 construct. Eighteen hours post-transfection, cells were serum starved for 24 hours. After the addition of 100 nM Insulin, whole cell lysates (WCL) were collected at the indicated time points for immunoblot analysis with the indicated antibodies, for Flag-IP and GST pull-down assays to determine Rictor/14-3-3 interaction, and for HA-IP to determine Rictor/Cullin-1 interaction.

G. HA-Cullin-1 expressing HeLa cells were treated with 20 μM LY294002. At the indicated time points, whole cell lysates (WCL) were collected for immunoblot analysis with the indicated antibodies, for HA-IP to determine Rictor/Cullin-1 interaction, and GST pull-down assays to determine Rictor/14-3-3 interaction.

(see also Figure S5)

To gain a better understanding of how phosphorylation of Rictor at T1135 functions physiologically in vivo, we used a stable HeLa cell line expressing HA-Cullin-1 at levels comparable to endogenous Cullin-1 (Figure S5I–J), and performed a series of time-course experiments to investigate how physiological manipulation of Rictor T1135 phosphorylation affects Cullin-1/Rictor interaction. As shown in Figure 5F, insulin addition into serum starved HeLa cells leads to activation of the S6K and Akt kinase pathways (as shown by the increased S473-Akt and T389-S6K signals), which results in increased Rictor phosphorylation at T1135, coupled with a decrease in Cullin-1/Rictor interaction and enhanced 14-3-3/Rictor interaction, and consequently, increased SGK1 abundance.

This data indicates that Rictor phosphorylation at T1135 might function in a similar fashion as the phospho-mimetic T1135E mutant in abolishing Rictor interaction with Cullin-1. However, T1135E failed to interact with 14-3-3 (Figure S5A and S5C), indicating that unlike Raptor phosphorylation by AMPK (Gwinn et al., 2008), simple recruitment of 14-3-3 after Rictor phosphorylation is not the only cause to disrupt Rictor/Cullin-1 interaction. We noticed that disruption of the endogenous interaction between 14-3-3 and Rictor by the R18 peptides leads to a sharp decrease in phosphorylation of Rictor at T1135 (Figure S5D). This indicates that 14-3-3 interaction might serve as a mechanism to protect T1135 from dephosphorylation, which has been described for Bad (Chiang et al., 2003; Datta et al., 1997) and Foxo3a (Singh et al.). In this sense, it is the phosphorylation event at Rictor T1135, rather than the interaction with 14-3-3 that results in disruption of the Cullin-1/Rictor complex. Although the T1135E Rictor mutant does not interact with 14-3-3, it behaves similarly as Rictor phosphorylated at T1135, both introducing a negative charge to cause a possible local conformation change to impair Rictor interaction with Cullin-1. In support of this, a peptide composed of 200 amino acids surrounding the T1135 site is sufficient to interact with Cullin-1 in vitro while the T1135E mutation leads to a significant reduction in their interaction in vitro (Figure 5E).

In a reciprocal set of experiments, we treated HA-Cullin-1 expressing cells cultured in 10% FBS-containing DMEM medium with LY290042 to inactivate PI 3-K and then monitored how this affects the Cullin-1/Rictor interaction. LY treatment induced a time-dependent decrease of Rictor T1135 phosphorylation, presumably due to the inactivation of S6K and Akt, which correlates with a decrease in 14-3-3 interaction and an increase in Rictor interaction with Cullin-1, resulting in decreased SGK1 abundance (Figure 5G). Therefore, in both experimental conditions, Rictor phosphorylation at T1135 is inversely correlated with its ability to interact with Cullin-1.

Phosphorylation of Rictor at T1135 reduces the ability of Rictor to ubiquitinate SGK1

As a result of reduced interaction with Cullin-1, T1135E Rictor is defective in promoting SGK1 ubiquitination (Figure 6A–C and S6A). Since T1135E has similar affinity as wild-type Rictor in binding to SGK1 (Figure S5H and S6B), the impaired E3 ligase activity towards ubiquitination of SGK1 might primarily be due to the disruption of the Cullin-1/Rictor association. Moreover, in keeping with impaired E3 ligase activity, compared with wild-type Rictor, T1135E Rictor is also compromised in promoting SGK1 degradation (Figure 6D and S6C), and thus is more potent at promoting S phase entry (Figure 6E). Consistent with the above findings, loss of the PTEN tumor suppressor, which activates the PI 3-K/Akt pathway, also results in elevated SGK1 expression (Figure S6D–E). This is directly correlated with increased Rictor phosphorylation at T1135. This argues that loss of PTEN may lead to elevated Akt and S6K activities, resulting in enhanced Rictor phosphorylation at T1135 and in turn, an impaired ability of the Rictor/Cullin-1 complex to degrade SGK1. In summary, these data demonstrate that in addition to complexing with mTOR to form the mTORC2 complex as a means to phosphorylate Akt and SGK, by complexing with Cullin-1 and Rbx1, Rictor might have an additional function as an E3 ligase complex that controls the stability of SGK1 and likely additional targets (Figure 7). We also show that the E3 ligase activity of Rictor is subject to negative regulation by a variety of AGC kinases. Since PI 3-K positively regulates AGC kinase activity, frequent hyperactivation of this signaling axis might contribute to the elevated SGK1 expression levels detected in various cancers (Figure 7).

Figure 6. Phosphorylation of Rictor at T1135 reduces the ability of Rictor to ubiquitinate SGK1.

Figure 6

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A. Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-HA immunoprecipitates of 293T cells transfected with the indicated plasmids. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight before harvesting.

B. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Δ60-SGK1 together with His-Ub and various Myc-Rictor constructs. Twenty hours post-transfection, cells were treated with the proteasome inhibitor MG132 overnight before harvesting. The whole cell lysates were collected in EDTA-free lysis buffer and the His-pull down was carried out in the presence of 8 M Urea to disrupt possible protein interactions.

C. Rictor/Cullin-1 promotes SGK1 ubiquitination in vitro. Immunopurified Cullin-1/Rictor complexes were incubated with purified recombinant SGK proteins (from Genway), purified E1, E2 and ubiquitin as indicated in 30°C for 45 minutes. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.

D-E. Wild type or Rictor−/− MEFs were transfected with the indicated Rictor plasmids (with empty vector as a negative control) together with the pBabe-Puro retroviral empty vector. Twenty-four hours post-transfection, the cells were treated with 1 μg/ml puromycin for 48–72 hours to kill the non-transfected cells prior to collecting the whole cell lysates for immunoblots (D), or subjected to BrdU analysis (E). Results are shown as means ± s.d. for three sets of experiments.

(see also Figure S6)

Figure 7.

Figure 7

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Proposed model for the Rictor/Cullin-1 pathway to control SGK1 turnover.

Discussion

The data presented above provides experimental evidence for a possible function of Rictor in the ubiquitination of SGK1. To date, the only function attributed to Rictor in the mTORC2 complex is phosphorylation of the hydrophobic motifs of downstream targets such as Akt and SGK. Our data points to a specific association of Rictor with Cullin-1 and Rbx1 as part of a possible functional E3 ubiquitin ligase complex (Figure 7).

Although our results indicate that mTOR may not be required for Rictor E3 ligase activity (Figure 2B), it remains unclear whether these two complexes share common scaffolding proteins and what the crosstalk is between these two complexes. Recent studies demonstrate that many other kinase complexes also possess E3 ligase activity (Lu et al., 2002; Maddika and Chen, 2009). Since many ubiquitination processes require a prior phosphorylation event, the coupling of both the kinase and the E3 ligase in proximity provides for more efficient destruction (Carrano et al., 1999). It is possible that there are unknown Rictor ubiquitination substrate(s) that require prior phosphorylation by Rictor.

Using gel filtration assays, in addition to the well-known mTORC2 complex migrating at over 600KD, we detected a second possible Rictor-containing complex, co-migrating with Cullin-1 and Rbx1 at 300–400KD (Figure S3G). This finding was supported by endogenous co-immunoprecipitation assays showing that only Rictor, but not other mTORC2 complex components including mTOR, GβL and Sin1 associates with Cullin-1 (Figure 3A–D and Figure S3A). We also found that although Sin1 could efficiently immunoprecipitate endogenous Rictor, it could not immunoprecipitate endogenous Cullin-1 as Rictor IP does (Figure 3C). Since Sin1 is required for formation of the mTORC2 complex (Jacinto et al., 2006; Yang et al., 2006), these results suggest that Rictor might exist in two distinct complexes, the Sin1-containing mTORC2 complex, and the Cullin-1-containing complex. This was further supported by the observation that depletion of Rictor, Cullin-1 and Rbx1, but not mTOR, Raptor or Sin1 leads to upregulation of SGK1 (Figure S1D and 3G). However, additional studies are required to fully understand the possible role of mTOR and other TORC2 complex components in Rictor-mediated SGK1 turnover.

Our data also demonstrate that unlike Rictor, Raptor does not promote the ubiquitination of Akt1, SGK1 and S6K1 (Figure 2B and S2B). This suggests that the E3 ligase activity might be unique to the Rictor/Cullin-1 complex. However, under ectopic expression conditions, Skp1 is not detected in the Rictor/Cullin-1 complex (Figure S3J-K). It is known that Skp1 serves as a bridging molecule to hold Cullin-1 and the F-box protein in a complex. The possible lack of Skp1 in the Cullin-1/Rictor complex suggests that Rictor might play a role equivalent to Skp1. However, further experimental investigation is required to fully understand the role of Skp1 for the Rictor/Cullin-1 complex to ubiquitinate SGK1, and more studies are also needed to determine whether a specific F-box protein is involved in this process.

Although SGK1 has been shown to be degraded by other E3 ligases including Nedd4-2 and CHIP, in both cases it is not known how the destruction is regulated and whether it is mediated by PI 3-K signaling. Our data suggest an alternative mechanism whereby multiple AGC kinases can negatively regulate Rictor/Cullin-1 E3 ligase activity without affecting its kinase activity, and this suggests a positive feedback loop to boost SGK1 activity post-stimulation. However, more studies are required to determine whether the ability of Nedd4-2 and CHIP to degrade SGK1 is also affected by the PI 3-K/Akt signaling. SGK1 overexpression has been reported in multiple cancers including breast cancer. Most interestingly, although SGK1 has been suggested to have redundant functions with Akt, simultaneous overexpression of both Akt and SGK1 has also been reported in breast cancers (Sahoo et al., 2005). Our work suggests that since SGK1, but not Akt, is subject to ubiquitination by the Rictor/Cullin-1 complex (Figure S6A), elevated Akt activity might block the function of Rictor/Cullin-1, leading to accumulation of SGK1 (data not shown and Figure S6F). Collectively, our results provide insight into how Rictor can influence SGK1 signaling by promoting its ubiquitination. Furthermore, we define a feedback mechanism that can negatively regulate the E3 ligase activity of the Rictor/Cullin-1 complex. This provides functional insight into Rictor regulation, as well as mechanistic information regarding the SGK1 stability controlled by the PI 3-K pathway, and how misregulation of this process contributes to SGK1 overexpression in human cancers.

Experimental Procedures

Cell culture and Cell Synchronization

Cell culture, including synchronization and transfection, has been described (Gao et al., 2009). Lentiviral shRNA virus packaging and subsequent infection of various cell lines were performed according to the protocol described previously (Boehm et al., 2005). Rictor −/− MEFs and control MEFs were kind gifts from Dr. Mark Magnuson (Shiota et al., 2006). Kinase inhibitors LY294002 (Sigma, L9908), Rapamycin (Calbiochem, 553210), Wortmannin (Sigma, 95455) and Akt 1/2 Inhibitor VIII (Calbiochem, 124018) were used as indicated.

In vitro Kinase Assay

mTORC2 in vitro kinase assay was performed as described previously (Sarbassov et al., 2005).

In vivo Ubiquitination Analysis

Cells were transfected with a plasmid encoding HA-Δ60-SGK1 along with Flag- or His-tagged ubiquitin. myc-Rictor or other expression vectors were co-transfected to assess their effects on SGK ubiquitination. Thirty-six hours after transfection, 10 μM MG132 was added to block proteasome degradation, and cells were harvested in EBC buffer or denaturing buffer (6 M Guanidine-HCL, 0.1 M NaH2PO4, 10 mM Tris-HCL, 10 mM immidazole, pH 8.0) containing protease inhibitor. 2 mg whole cell lysates were incubated with Flag beads or Ni-NTA resin for 4–10 hrs, followed by washing 4 times with NETN buffer or denaturing washing buffer (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCL, 10 mM immidazole, pH 6.3). Then the washed pellet was boiled in SDS-containing lysis buffer and resolved on SDS-PAGE.

In vitro Ubiquitination Assay

The in vitro ubiquitination assays were performed as described previously (Jin et al., 2005). To purify the Cullin-1/Rictor E3 ligase complex, 293T cells were transfected with vectors encoding HA-Cullin-1, Myc-Rictor (WT or T1135E), and Flag-Rbx1. The Cullin-1/Rictor (E3) complexes were purified from the whole cell lysates using HA-agarose beads. Purified, recombinant SGK protein (purchased from Genway) were incubated with purified Cullin-1/Rictor (E3) complexes in the presence of purified, recombinant active E1, E2 (UbcH5a and UbcH3), ATP and ubiquitin. The reactions were stopped by the addition of 2X SDS-PAGE sample buffer and the reaction products were resolved by SDS-PAGE gel and probed with the indicated antibodies.

Mass Spectrometry analysis to detect Rictor T1135 phosphorylation in vivo

293T cells were transiently transfected with Myc-Rictor plasmid, and eighteen hours post-transfection, cells were grown in serum deprived conditions for 24 hours. Thirty minutes after addition of insulin, whole cell lysates were collected to perform Myc-immunoprecipitation. Myc-immunoprecipitates were resolved on SDS-PAGE and visualized by colloidal Coomassie Blue. The band containing Rictor was excised and treated with DTT to reduce disulfide bonds and iodoacetamide to derivatize cysteine residues. In-gel digest of the protein was performed with trypsin. The peptides were extracted from the gel and phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC), then analyzed by nanoscale-microcapillary reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) essentially as described previously (Villen and Gygi, 2008). Peptides were separated across a 37-min gradient ranging from 4% to 27% (v/v) acetonitrile in 0.1% (v/v) formic acid in a microcapillary (125 μm ×18 cm) column packed with C18 reverse-phase material (Magic C18AQ, 5 μm particles, 200 Å pore size, Michrom Bioresources) and on-line analyzed on The LTQ Orbitrap XL™ hybrid FTMS (Thermo Scientific, Bremen, Germany). For each cycle, one full MS scan acquired on the Orbitrap at high mass resolution was followed by ten MS/MS spectra on the linear ion trap XL from the ten most abundant ions. MS/MS spectra were searched using the SEQUEST algorithm (Eng et al., 1994) against database created on the basis of a protein sequence database containing the sequence of Rictor, of common contaminants, such as human keratin proteins with static modification of cysteine carboxymethylation, dynamic modification of methionine oxidation and serine, threonine and tyrosine phosphorylation. All peptide matches were filtered based on mass deviation, tryptic state, XCorr and dCn and confirmed by manual validation. The reliability of site-localization of phosphorylation events was evaluated using the Ascore algorithm (Beausoleil et al., 2006).

Gel Filtration Chromatography for Separation of Rictor Complexes

Two 10 cm plates of HeLa cells were washed with phosphate buffered saline, lysed in 0.5 ml of CHAPS lysis buffer (25 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 0.3% CHAPS), and filtered through a 0.45 μm syringe filter. Total protein concentration was 6 mg/ml. 500 μl of lysate was loaded onto a Superdex 200 10/300 GL column (GE Lifesciences Cat. No. 17-5175-01) (Figure S3G). The experiment in Figure S5E was performed with this same column attached in series to a Superose 6 10/300 GL (GE Lifesciences Cat. No. 17-5172-01). The sample was separated in the Superose 6 column first, and then the Superdex 200 column. The gel filtration beads in each column have different size exclusion characteristics that complement each other and allow separation of very large and also smaller proteins. Chromatography was performed using an AKTAFPLC (GE Lifesciences Cat. No. 18-1900-26) and protein complexes were resolved by eluting with the same CHAPS buffer at 0.5 ml/min over 3 hours. Eluent was collected in either 250 μl (Figure S3G) or 500 μl (Figure S5E) fractions. 50 μl aliquots of these fractions were loaded onto SDS-PAGE minigels for SDS-PAGE and western blot analysis of the fractionated protein complexes. Prior to running cell lysates, the molecular weight resolution of the columns was first estimated by running native molecular weight markers (urease ~545, mouse monoclonal IgG ~180 kDa, human serum albumin ~68 kDa) and determining their retention times on coomassie-stained SDS-PAGE protein gels.

Supplementary Material

01

Acknowledgments

We thank Lewis Cantley, Qing Zhang, Christoph Schorl, Ben Liu and Susan Glueck for critical reading of the manuscript; Christoph Geisen, Brendan Manning, John Blenis, Yue Xiong, Bing Su, Suzanne Conzan and Mark Magnuson for providing reagents; Brendan Manning for sharing unpublished data, and members of the Wei, DeCaprio, Harper and Toker labs for useful discussions. Wenyi Wei is a Kimmel Scholar, V Scholar and Karin Grunebaum Cancer Research Foundation Fellow. This work was supported in part by the DOD Prostate New Investigator award to W.W., and by grants from the National Institutes of Health (A.T., CA122099; W.W., GM089763).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental information includes six figures and Supplemental Experimental Procedures, and can be found with this article online.

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