Tuberin localization is regulated by Ser/Thr phosphorylation in response to growth factor stimulation
To investigate the subcellular localization of tuberin, mouse fibroblast (NIH3T3, Swiss3T3), rat kidney epithelial (TRKE2), human embryonic kidney (HEK293), and human breast cancer (MCF7) cell lines were fractionated, revealing that tuberin was detected within both the cytosolic and membrane fractions but not within the nuclear fraction (). Interestingly, tuberin in the membrane fraction migrated faster than tuberin within the cytosolic fraction ().
Figure 1. Tuberin localization is determined by Ser/Thr phosphorylation. (A) Western analyses of subcellular fractions from the indicated cell lines using an anti-tuberin antibody. β1-integrin and lamin A/C proteins were analyzed as fractionation controls. (more ...)
To determine whether this mobility shift was due to changes in phosphorylation, we treated these subcellular fractions with either a Ser/Thr or Tyr phosphatase (calf intestinal alkaline phosphatase [CIAP] or YOP protein tyrosine phosphatase, respectively). After CIAP treatment, the mobility of tuberin in the cytosol increased, resolving as a faster migrating band similar to tuberin purified from membrane fractions (). However, YOP did not affect tuberin mobility, indicating that the mobility shift was primarily due to phosphorylation of Ser/Thr residues. Treatment with CIAP resulted in both membrane and cytosolic tuberin migrating faster, indicating that tuberin within the cytosolic fraction is hyperphosphorylated. In addition, the serum-induced decrease in mobility of tuberin in both membrane and cytosolic fractions is likely driven by multiple phosphorylation events, as CIAP treatment (removing all Ser/Thr phosphorylation) increased the mobility of tuberin in both fractions.
To determine whether growth factor stimulation could also alter tuberin phosphorylation and subcellular localization, Swiss3T3 or MCF7 cells were serum starved and then stimulated with serum or insulin-like growth factor-1 (IGF-1) before fractionation (; and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200507119/DC1
), as were NIH3T3 (Fig. S1 A) and TRKE2 cells (Fig. S1 B). Like serum, IGF-1 increased the amount of tuberin in the cytosolic fraction relative to starvation conditions (). In serum-stimulated cells, phosphorylation of membrane-localized tuberin was also increased after 1 h (comparable to cytosolic tuberin in starved cells but less than cytosolic tuberin from serum- stimulated cells; ), suggesting that phosphorylation at specific residues, rather than total levels of phosphorylation, was determining localization. By 6 h, tuberin became predominantly membrane localized, which correlated with a decrease in AKT activation (). Translocation of tuberin from the membrane to the cytosol was blocked by the PI3K inhibitors wortmannin and LY294002 ( and Fig. S1 B), implicating PI3K signaling in tuberin localization to the cytosol.
Tuberin translocation is mediated by AKT phosphorylation
To determine whether AKT directed tuberin's subcellular localization, tuberin from membrane and cytosolic compartments of NIH3T3 cells treated with IGF-1 or EGF was immunoprecipitated and detected with a (S/T) phosphosubstrate antibody that recognizes the consensus phosphorylation site for AKT and RSK containing phospho-Ser/Thr with Arg at position −5 and −3 (RXRXXpS/T) (Alessi et al., 1996
; Obata et al., 2000
; Yaffe et al., 2001
; Roux et al., 2004
). With equal tuberin loading (), the (S/T) phosphosubstrate antibody predominantly recognized tuberin within the cytosolic fraction, and the PI3K inhibitor wortmannin significantly reduced recognition of phosphorylated tuberin, suggesting that cytosolic tuberin was phosphorylated by AKT (). To confirm that activation of AKT directed tuberin to the cytosol, MCF7 cells stably transfected with a constitutively active AKT (myr-AKT) were examined and found to contain more cytosolic tuberin relative to wild-type MCF7 cells (). Recently, RSK was shown to phosphorylate tuberin at S1798 (Roux et al., 2004
). To determine whether RSK phosphorylation of tuberin could regulate its localization, we generated a COOH-terminal deletion mutant of TSC2 at residue 1734 (referred to as Δ73) that lacks S1798 (Roux et al., 2004
). Δ73 and wild-type tuberin had a similar distribution within membrane and cytosolic fractions under normal growth conditions (Fig. S1 C) and in response to serum (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200507119/DC1
). In addition, overexpression of RSK1 did not affect localization of wild-type tuberin (Fig. S2 B). Collectively, these data indicate that tuberin residing in the cytosol is phosphorylated by AKT, suggesting that AKT (but not RSK) directly controls tuberin's localization and possibly its activity.
Figure 2. AKT-mediated phosphorylation of tuberin leads to subcellular translocation. (A) NIH3T3 cells were treated with 100 ng/ml EGF in the absence or presence of 200 nM wortmannin for 1 h. The fractionated cell lysates were immunoprecipitated with an anti-tuberin (more ...)
Mutation of AKT phosphorylation sites in tuberin alters its localization
Tuberin contains multiple S/T phosphorylation sites (). Among these, S1254 has been shown to be phosphorylated by MK2 (Li et al., 2003
), whereas S939 (Inoki et al., 2002
; Manning et al., 2002
), S981 (Dan et al., 2002
), S1130/S1132 (Inoki et al., 2002
), and T1462 (Inoki et al., 2002
; Manning et al., 2002
) have been reported to be AKT phosphorylation sites in vivo and/or in vitro, and several of these also have the potential for binding to 14-3-3 (http://scansite.mit.edu
; Yaffe et al., 2001
). S981 is of particular interest, as it lies within the alternatively spliced exon 25 of tuberin. To examine these as candidate sites for the regulation of tuberin localization, we mutated these residues to alanine to create TSC2 constructs in frame with an NH2
-terminal Flag epitope (Flag-TSC2): S939A, S981A, and 2A (S939A + S981A); T1462A and SATA (S939A + T1462A; Manning et al., 2002
); and S1254A and S1130A + S1132A double mutant.
Figure 3. Identification of S939 and S981 as phosphorylation sites that determine tuberin localization. (A) Schematic of tuberin phosphorylation sites with Flag-TSC2 constructs and their corresponding mutations listed below. (B) Western analysis of HEK293 membrane (more ...)
Wild-type and mutant TSC2 constructs were transfected into HEK293 cells and subjected to subcellular fractionation. As shown in , S939A, S981A, and 2A mutants predominantly localized to the membrane, as did the double alanine SATA mutant that lacks the S939 site. However, the T1462A single mutant partitioned in the cell similarly to wild-type tuberin (), indicating that T1462 phosphorylation was not directing translocation of tuberin from the membrane to the cytosol. These data were confirmed with a phosphospecific T1462 antibody, which recognized tuberin in both the membrane and cytosolic fractions equally (unpublished data). Furthermore, phosphorylation at both S939 and S981 contributes to cytosolic localization, as phosphorylation at S939 (determined with a phospho-S939 specific antibody) of the S981A mutant was not sufficient to partition tuberin to the cytosol (Fig. S2 C). Other tuberin mutants, S1254A, S1130A/S1132A (), Δ73, or S1338A (Fig. S1 C), also distributed equally between the membrane and cytosolic fractions. Thus, S939 and S981 phosphorylation are critical determinants of membrane versus cytosolic localization of tuberin.
14-3-3 proteins mediate translocation of tuberin into the cytosol
14-3-3 has been previously reported to directly interact with phosphorylated tuberin (Li et al., 2002
; Nellist et al., 2003
). S939 and S981 are predicted AKT phosphorylation and 14-3-3 interaction sites. When phosphorylated and nonphosphorylated S939 and S981 peptides were used in competition assays to block GST–14-3-3 interaction with tuberin, as shown in and Fig. S3 (available at http://www.jcb.org/cgi/content/full/jcb.200507119/DC1
), phosphorylated but not nonphosphorylated S939 and S981 peptides clearly competed for the interaction of tuberin with several 14-3-3 isoforms. In addition, the amount of 2A mutant tuberin affinity purified by 14-3-3 was dramatically reduced relative to wild-type tuberin (). Importantly, inhibition of PI3K signaling by wortmannin ablated this 14-3-3 tuberin interaction (). These data indicate that S939 and S981 are critical sites of interaction between tuberin and 14-3-3 proteins and that this interaction is mediated by PI3K/AKT phosphorylation.
Figure 4. 14-3-3 proteins bind and sequester tuberin in the cytosol. (A) GST–14-3-3 proteins were used to affinity purify proteins from HEK293 cells in the presence of phosphorylated or nonphosphorylated S939 and S981 tuberin peptides. Affinity-purified (more ...)
To demonstrate that tuberin binding to 14-3-3 was directly responsible for its translocation to the cytosol, we transfected HEK293 cells with the EGFP-R18 construct that expresses a peptide that disrupts 14-3-3 binding (Jin et al., 2004
). As shown in , the R18 14-3-3 decoy clearly repressed cytosolic localization of tuberin, establishing a direct link between 14-3-3 and translocation of tuberin to the cytosol.
Hamartin enhances tuberin retention at the membrane
Hamartin possesses a predicted transmembrane domain and two coiled-coil domains that mediate its association with tuberin (van Slegtenhorst et al., 1997
). We found that in both human and mouse cells, hamartin was only detected in the membrane fraction (; ; unpublished data). To confirm that tuberin mutants that did or did not constitutively localize to the membrane retained their ability to bind hamartin, immunoprecipitation experiments were performed. Immunoprecipitation showed that, similar to wild-type tuberin, tuberin S939A, S981A, S1338A, Δ73, and 2A mutants retained their ability to interact with hamartin (Fig. S4, A, B, and C, available at http://www.jcb.org/cgi/content/full/jcb.200507119/DC1
). To determine whether hamartin played a role in the subcellular localization of tuberin, we transfected wild-type, S939A, and S981A Flag-TSC2 constructs into HEK293 cells with or without Myc- or Flag-TSC1. Although S939A and S981A mutants were primarily membrane localized, coexpression of hamartin increased membrane retention of both mutant and wild-type tuberin in a dose-dependent manner (). These data suggest that the interaction of hamartin with tuberin facilitates its localization to the membrane, implying that the tuberin–hamartin heterodimer functions in this subcellular compartment.
Figure 5. Hamartin increases the amount of tuberin retained in the membrane. (A) HEK293 cells were transfected with wild-type and mutant Flag-TSC2 constructs in the presence or absence of a Myc-TSC1 construct. Cells were fractionated and immunoblotted with indicated (more ...)
Importantly, only tuberin in the membrane fraction remained associated with hamartin: no hamartin could be coimmunoprecipitated with tuberin in the cytosolic fraction, indicating that translocation to the cytosol dissociated tuberin from hamartin (). Similarly, when Flag-TSC2 and Myc-TSC1 were cotransfected into MCF7 cells, tuberin and hamartin were observed by confocal microscopy to colocalize in a discrete, punctate pattern (Fig. S4 D). Cells that expressed wild-type Flag-TSC2 in the absence of Myc-TSC1 exhibited a more diffuse staining pattern than cells that overexpressed both Myc-TSC1 and Flag-TSC2 (Fig. S4 D, comparing γ and γ′ with β and β′). However, the 2A mutant, which was constitutively membrane localized as shown by cell fractionation, retained this punctate localization pattern even in the absence of exogenously expressed hamartin (Fig. S4 E).
Colocalization of tuberin and Rheb is disrupted in response to growth factor stimulation
Tuberin's GAP target Rheb is farnesylated and predicted to be membrane localized (Clark et al., 1997
), and recent data indicate that Rheb is localized in endomembranes (Takahashi et al., 2005
). To determine whether phosphorylation at S939/S981 affected tuberin's intrinsic GAP activity for Rheb, we first determined Rheb's subcellular localization. Rheb was detected only in the membrane fractions from HEK293 () and MCF7 (Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200507119/DC1
) as well as NIH3T3, Swiss3T3, and TRKE2 cells (not depicted). Treatment with EGF or wortmannin did not affect this localization over a period of 10 min to 3 h (Fig. S5 A and not depicted). Recognition of the 21-kD band in the membrane fraction using this anti-Rheb antibody was specific for Rheb, as antibody binding was ablated when Rheb RNAi was used to knock down Rheb (Fig. S5 B). When untagged TSC1
were cotransfected into HeLa cells, tuberin and Rheb were observed with confocal microscopy to colocalize in a discrete, punctate pattern in the absence of serum (). In contrast, stimulation with IGF-1 resulted in partitioning of wild-type tuberin away from Rheb (), indicating that in response to growth factor stimulation, tuberin is no longer retained in physical proximity to its downstream target, Rheb. Collectively, these data indicate that the localization pattern of tuberin is modulated by both hamartin and growth factor signaling and that Rheb is in physical proximity to membrane-localized tuberin and hamartin within the cell. In addition, these data, along with confocal microscopy using 2A mutant tuberin and biochemical fractionation, suggest that S939 and S981 are crucial sites for determining the subcellular localization and, thus, function of tuberin in response to growth factor signaling.
Figure 6. Tuberin colocalization with Rheb is disrupted in response to growth factor stimulation. HeLa cells were transfected in serum-free media with Flag-TSC2 WT and Myc-Rheb along with an untagged TSC1 construct. After 12 h without serum, cells were treated (more ...)
Tuberin localization regulates its function
To elucidate the relationship between tuberin localization and function, we examined the effect of wild-type, S939A, S981A, and 2A tuberin mutants on mTOR-S6K activity. T389 is a known rapamycin-sensitive phosphorylation site of S6K that correlates with activation by mTOR (Pearson et al., 1995
). In cells transfected with HA-tagged S6K, cotransfection of wild-type TSC1
expression constructs diminished phosphorylation of exogenously expressed S6K (). However, cotransfection of the 2A mutant with TSC1
resulted in an even more dramatic reduction in phospho-S6K levels (). Similar experiments were performed to determine the effect of S939A and S981A single and double mutants on phosphorylation of endogenous S6K. In the presence of serum, wild-type Flag-tuberin was located in the membrane and cytosol, whereas S939A, S981A, and 2A Flag-tuberin mutants remained primarily at the membrane (, top). AKT was equally activated by serum in cells expressing wild-type or mutant tuberin constructs, and endogenous hamartin remained membrane localized (). In cells transfected with wild-type tuberin, phosphorylation of endogenous S6K at T389 increased in response to serum, coincident with translocation of tuberin to the cytosol. However, in cells transfected with S939A, S981A, or 2A tuberin mutants, phosphorylation of S6K was inhibited, with the 2A mutant most effectively blocking S6K activation (). Thus, tuberin mutants that were retained at the membrane were constitutively active and exhibited enhanced ability to repress S6K activation. These data suggest that AKT phosphorylation of tuberin regulates its activity by decreasing the amount of tuberin located at the membrane, thereby reducing its inhibitory effect on the Rheb–mTOR–S6K signaling pathway.
Figure 7. AKT-mediated phosphorylation of tuberin relieves repression of mTOR-S6K signaling. (A) HEK293 cells were transfected with HA-S6K, Myc-TSC1, and Flag-TSC2 constructs as indicated. Cells were serum starved for 24 h and treated with or without 30 ng/ml IGF-1 (more ...)
Phosphorylation sites that determine localization and function of tuberin do not change its intrinsic RhebGAP activity in vitro
Physical sequestration of tuberin from its target Rheb suggested a mechanism whereby AKT modulated tuberin by partitioning it away from the membrane rather than altering its intrinsic GAP activity for Rheb. We compared the relative RhebGAP activity of wild-type TSC2 and 2A mutant (S939A and S981A) in cells during activation of the PI3K–AKT pathway (). To do this, we quantified the ratio of GTP and GDP Myc-Rheb (percentage of GTP bound Myc-Rheb) when these TSC2 constructs were coexpressed during a time course of insulin stimulation. The 2A mutant enhanced the GTPase function of Rheb more effectively than wild-type tuberin, as indicated by impaired accumulation of the active GTP form of Rheb after 15 min of insulin stimulation (32% GTP bound) when compared with wild-type TSC2 (42% GTP bound; ). As an increase in GTP bound Rheb would be predicted to enhance mTOR-mediated cell signaling, we measured the activity of HA-S6K1 in these cells (). As expected, insulin-induced activation of S6K1 was markedly impaired in cells expressing the 2A mutant with the 2A mutant blocking insulin-induced activation of S6K1 by 50% (after 15 min) when compared with wild-type TSC2 (), reflecting the reduced levels of active GTP bound Rheb ().
Figure 8. AKT phosphorylation of tuberin promotes Rheb-induced S6K1 activation through increased Rheb-GTP loading. (A) HEK293 cells coexpressing HA-S6K1, Myc-Rheb, Flag-TSC1, and either Flag-TSC2-WT or Flag-TSC2-2A (S939A and S981A) were serum starved and, where (more ...)
To determine whether phosphorylation at S939 and S981 had an impact on tuberin's intrinsic GAP activity, we analyzed the ability of wild-type or mutant tuberin proteins to activate Rheb GTPase in vitro. RhebGAP assays were conducted on immunoprecipitated tuberin–hamartin heterodimers containing either the wild-type or AKT-phosphorylation mutants of TSC2 (). Tuberin–hamartin complexes containing wild-type tuberin from both unstimulated and insulin-stimulated cells enhanced the intrinsic GTPase activity of Rheb at comparable rates. Induction of tuberin phosphorylation by insulin was confirmed in these lysates by detection of phospho-T1462 (, bottom). Furthermore, tuberin mutants lacking these sites (S939A, S981A, and 2A) also possessed similar RhebGAP activity to wild-type tuberin in vitro, even after insulin treatment (). These findings imply that AKT-mediated phosphorylation of tuberin does not directly alter the rates at which tuberin enhances the GTPase activity of Rheb, at least in vitro. However, these data support a mechanism whereby AKT phosphorylation suppresses tuberin function by translocating tuberin to the cytosol away from its membrane-associated binding partner, hamartin, and its downstream target, Rheb. Indeed, as the efficiency with which tuberin functions as a RhebGAP in vitro is significantly reduced in the absence of hamartin, physical separation from both hamartin and Rheb may be contributing to reduced tuberin activity (Tee et al., 2002
; Li et al., 2004b
Figure 9. Tuberin's GAP activity for Rheb in vitro is not affected by AKT phosphorylation. HEK293 cells coexpressing Flag-TSC1 and Flag-TSC2-WT, Flag-TSC2-S939A, Flag-TSC2-S981A, or Flag-TSC2-2A were serum starved and then stimulated with 100 nM insulin for 30 (more ...)
A mechanism by which tuberin function is regulated through subcellular localization
Based on our data, we propose a model where in the absence of growth stimulatory signals, hamartin facilitates localization of hypophosphorylated tuberin to membranes in physical proximity to Rheb. The membrane-associated tuberin–hamartin complex binds Rheb and acts as a GAP to inactivate membrane Rheb signaling by stimulating GTP hydrolysis (). However, during mitogenic sufficiency (i.e., after growth factor stimulation), activation of PI3K signaling leads to activation of AKT, which then directly phosphorylates membrane-associated tuberin. In response to AKT phosphorylation, 14-3-3 proteins bind tuberin and sequester it in the cytosol. The deficiency of membrane-associated tuberin results in the accumulation of GTP bound Rheb, which leads to increased mTOR signaling, enhanced cell growth, and proliferation ().
Figure 10. Model for regulation of tuberin under conditions of mitogenic sufficiency. (A) Tuberin normally functions as a GAP for Rheb within an intracellular membrane compartment, inhibiting Rheb and mTOR signaling, which suppresses cell growth. (B) Upon stimulation (more ...)