Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Luisa Beltran; Claire Chaussade; Bart Vanhaesebroeck; Pedro Rodriguez Cutillas

doi:10.1073/pnas.1107692108

Edited by Jonathan R. Hart, The Scripps Research Institute, La Jolla, CA, and accepted by the Editorial Board August 9, 2011 (received for review May 20, 2011)

Abstract

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein–protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity.

Class IA phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinase signaling enzymes that act downstream of Ras, tyrosine kinase receptors, and G-protein coupled receptors. They control key cellular functions, including survival, growth, proliferation, migration, and metabolism, many of which are regulated through the downstream effector Akt (also known as protein kinase B) (1 –3).

PI3K signaling must be tightly regulated in cells to ensure efficient metabolism, survival, and orderly growth. The importance of appropriate regulation of this pathway is illustrated by the observation that malfunction of the PI3K/Akt pathway can lead to conditions as diverse as the metabolic syndrome, autoimmune diseases, neurodegeneration, and cancer (4 –8). Cells have therefore evolved multiple mechanisms to regulate the activity of the PI3K/Akt axis. The most well known of these involves the activities of lipid phosphatases, such as phosphatase and tensin homolog (PTEN), which dephosphorylate phosphatidylinositol (3,4,5)-triphosphate, the lipid product of class IA PI3K, to produce phosphatidylinositol (4,5)-biphosphate (9). Inactivation of PTEN, as a mechanism to evade PI3K pathway control, is a common occurrence in cancer (9). The pathway is also regulated by protein phosphatases, such as protein phosphatase 2A (PP2A), which dephosphorylate and inactivate Akt (10), and by negative feedback loops that terminate the activation signal, as exemplified by the phosphorylation and subsequent degradation of insulin receptor substrate 1 by mammalian target of rapamycin complex 1 (mTORC1) (11).

Signaling activity is often regulated by the formation of multiprotein complexes that serve to modulate enzymatic activity allosterically and/or to position proteins in their appropriate subcellular location (12, 13). Regulation of signaling processes by protein–protein interactions is gradually being elucidated for several signaling nodes including ERK (14), Akt (15), and mTOR (16, 17), to cite some examples. However, the extent by which protein–protein interactions may modulate the activity of PI3K enzymes is currently underexplored. It is known that binding of p85 to p110 leads to a decrease in the lipid kinase activity of p110 in vitro (18). However, whether p85 regulates p110 activity dynamically in vivo is not clear as this interaction is very strong and both subunits are constitutively bound in cells (19). Ras binding to the p110α subunit was deduced by structural analysis and, although this interaction is known to be critical for some PI3K functions (20, 21), it does not survive biochemical purification, indicating that it is a weak and transient interaction. A number of other proteins have been reported to bind p85 including SH2 domain-containing inositol 5' phosphatase (22), ezrin (23), Src family members (24), and the PTEN-associated complex (25). However, the potential roles, if any, of these binding partners in the regulation of PI3K have not been investigated extensively.

Here, we aimed to identify previously unknown PI3K interaction partners with a functional role in controlling the activity of the pathway. We reasoned that binding partners with an active function in modulating signaling would bind PI3K at different stoichiometries in cells with an active pathway relative to those in which the pathway was inactive. An affinity purification mass spectrometry (AP-MS) strategy (Fig. 1A), inspired by that described for the identification of epidermal growth factor receptor signalosomes (26), was designed to quantify binding partners of class IA PI3K in starved cells relative to cells treated with serum. These analyses identified calpain small subunit 1 (CAPNS1) as a unique dynamic interaction partner of PI3K that was preferentially in complex with p85 in starved cells. Subsequent investigations confirmed that enzymatically active calpain associated with the PI3K protein complex and that this association correlated with PI3K activity. Furthermore, calpain 1 and calpain 2 were found to cleave the p110α isoform of PI3K and to reduce its PI3K lipid kinase activity. Subsequent functional experiments revealed that calpains have a negative role in the regulation of PI3K/Akt signaling and that their inhibition promotes cell viability during serum starvation.

Fig. 1.

AP-MS screen for interaction partners of PI3K. (A) Scheme of AP-MS strategy. (B) Volcano plot illustrating relative quantification of proteins identified in all three experiments (n = 81). (C) Representative MS/MS spectrum of a tryptic peptide derived from CAPNS1. (D) Extracted ion chromatograms (XICs) for the same representative CAPNS1 peptide in IPs obtained from starved or serum-stimulated cells. (E) Relative protein quantifications of CAPNS1 and PI3K subunits in serum-starved cells before and after serum stimulation. Relative protein abundance was expressed as mean ± SEM (n = 3 independent experiments) relative to basal sample. **, p < 0.01; ns, not significant.

Results

AP-MS Study Suggests That CAPNS1 Interacts with PI3K in NIH 3T3 Cells as a Function of Pathway Activation.

We used a quantitative AP-MS strategy to identify interaction partners of PI3K (Fig. 1A). Protein complexes were immunoprecipitated from approximately 8 × 10⁶ NIH 3T3 cells using a mixture of two antibodies against the p85 regulatory subunit of PI3K (antibodies raised against the N-terminal SH2 domain and full-length p85). Mild cell lysis conditions (0.3% CHAPS) were used to maximize the chances of retaining weak protein–protein interactions. This approach was selected because we wished to investigate interaction partners of endogenous PI3K without the need to overexpress the protein bait. Purified PI3K protein complexes were then fractionated using 1D SDS-PAGE and subjected to in-gel trypsin digestion prior to liquid chromatography (LC)-MS and LC-MS/MS.

PI3K protein complexes were purified and analyzed from cells that were serum starved for 18 h (“starved”) and from cells that were serum starved for 18 h followed by a 5-min stimulation with 10% FBS (“stimulated”), resulting in low and high PI3K/Akt pathway activity, respectively. A quantitative approach was taken to identify candidate binding partners of PI3K. Proteins present in equal abundance in immunoprecipitates (IPs) under both conditions were considered to be nonspecific or constitutive interactors and were not investigated further. We focused our attention on proteins that interacted dynamically with PI3K upon serum-induced pathway activation because we considered that they may have potential functional roles in the regulation of PI3K signaling. Three biologically independent experiments were performed.

Hundreds of proteins were identified and quantified across the study, with an average of 193 proteins found per experiment. Of these, 81 proteins were identified and quantified in all three replicates (Table S1). We used rigorous criteria for both protein identification and quantification to identify potential dynamic binding partners of PI3K: First, proteins had to be identified in IPs with a minimum of three peptides and a MASCOT score of 40; second, proteins had to be identified and quantified across the three independent replicates; third, the average log ₂-fold difference of protein abundance in IPs obtained from starved and stimulated cells had to be < -1 or > 1 (i.e., twofold); fourth, the quantitative difference had to be statistically significant (p < 0.05 as assessed by the Student t test). Three proteins met these criteria (highlighted in Fig. 1B). Of these, CAPNS1 was identified in PI3K protein complexes in all three experiments (Fig. 1C and Table S2) and was found at lower abundance in serum-stimulated cells relative to starved cells, with a mean log ₂-fold difference of 1.45 (SD = 0.31; p = 0.010) (Fig. 1 D and E). The PI3K subunits p85α, p85β, p110α, and p110β were also identified and quantified in all three experiments and were found to be present in equal abundance under both conditions (Fig. 1E and Table S2).

Calpains are a family of calcium-dependent cysteine proteases (27), of which the ubiquitously expressed isoforms calpain 1 and calpain 2 are the most extensively studied. Both isoforms are heterodimers comprised of a common 28-kDa regulatory subunit and an 80-kDa catalytically active subunit (27). The activation and regulation of calpain activity in vivo is not well understood, although it is an abundant protein with limited proteolytic activity and therefore it is assumed to be tightly regulated (28). Calpains are thought to cleave proteins at highly specific recognition sites, although no consensus cleavage sequence has been identified. Instead, secondary structural features may be more important recognition factors for the protease (27, 29). Calpain activity most frequently results in the production of large polypeptide fragments, suggesting that it is more likely to have a role in regulating substrate activity than in substrate digestion (29, 30). Reflecting the diversity of their substrates, calpains have been implicated in the regulation of multiple biological processes including apoptosis, autophagy, proliferation, and migration (28, 31).

Calpain and PI3K Interact Dynamically.

Given the important role of calpains in cell signaling, we next sought to confirm the interaction between PI3K and calpain using independent methods. Because of the sensitivity limitations of the available antibodies against calpains for immunoblotting, published studies have used activity assays to investigate calpain expression in cells (32, 33). We thus pursued this strategy to test the PI3K–calpain interaction further. PI3K was immunoprecipitated from starved and serum-stimulated NIH 3T3 cells using antibodies against the p85 regulatory subunit of PI3K. Total cell lysate (TCL) and PI3K IPs were then subjected to an in vitro assay for calpain protease activity (Fig. 2A). Calpain activity was detectable in both TCL and p85 IPs. Calpain activity levels were not found to be significantly different in starved or stimulated NIH 3T3 TCL (Fig. 2A). In contrast, calpain activity in p85 IPs was found to be decreased upon serum stimulation (Fig. 2A).

Fig. 2.

Confirmation that the calpain–PI3K interaction is dynamically regulated. (A) Total cell lysates (n = 3) and purified protein complexes (n = 4) were subjected to an in vitro protease assay for calpain activity after serum starvation for 18 h and serum stimulation for the indicated times. *below, below limit of assay detection; ns, not significant; *, p < 0.05. An in vitro lipid kinase assay was used to detect PI3K activity in calpain immunoprecipitates from (B) cells serum starved for 18 h followed by serum stimulation as indicated (n = 3) and (C) cells serum starved for 8 or 18 h (n = 2). **, p < 0.01.

To investigate the PI3K–calpain interaction further, calpain was immunoprecipitated from starved and serum-stimulated NIH 3T3 cells using antibodies against calpain catalytic subunit proteins, including calpain 1 and calpain 2. Purified proteins were then subjected to an in vitro lipid kinase assay for PI3K activity (34). PI3K activity was detectable in calpain IPs from cells serum starved for 18 h and was found to be reduced by approximately 60% upon serum stimulation (Fig. 2B). Furthermore, PI3K activity in calpain protein complexes was found to be higher after 18 h serum starvation compared to 8 h serum starvation (Fig. 2C). Thus, in line with observations from the AP-MS study (Fig. 1), these results indicate that the interaction between calpain and PI3K is enhanced in response to serum starvation and decreased upon cell stimulation that leads to an activation of PI3K signaling.

Calpain 1 and 2 Cleave p110α and p85 and Reduce PI3K Enzymatic Activity.

We next determined the effects of calpain activity on PI3K protein stability and activity. PI3K proteins were immunoprecipitated from NIH 3T3 cells using antibodies against the p85 regulatory subunit and used as substrate for an in vitro calpain activity assay. Purified calpain 1 and calpain 2 proteins were used as the protease source. Western blot (WB) analysis revealed that calpain 1 and calpain 2 cleaved p110α completely, whereas p85 was partially cleaved by both calpain 1 and calpain 2 (Fig. 3A). Conversely, the effect of calpain activity on the stability of p110β was negligible (Fig. 3A). These results demonstrate that calpain can cleave PI3K proteins in vitro and suggest that calpain may have a role in p110α stability in vivo.

Fig. 3.

Effects of calpain activity on PI3K stability and lipid kinase activity. PI3K subunits were immunoprecipitated from NIH 3T3 cells and used as substrates for an in vitro protease digestion using purified calpain isoforms 1 and 2. (A) WB analysis of digested PI3K subunits (one representative experiment of three) and (B) lipid kinase assay for PI3K activity of digested PI3K subunits (n = 4). **, p < 0.01. (C) Levels of p85, p110α, and p110β were measured by WB in NIH 3T3 cells subjected to siRNA knockdown for 48 h using CAPNS1 or scrambled siRNA followed by 18 h serum starvation (n = 3). A representative experiment and mean ± SEM (normalized to α-tubulin) are shown in top and bottom panels, respectively. *, p < 0.05.

The activity of calpain-cleaved PI3K proteins was evaluated by immunoprecipitating PI3K proteins (using antibodies against p85) from NIH 3T3 cells and subjecting them to an in vitro protease digestion using purified calpain, followed by a lipid kinase assay for PI3K activity. Results demonstrated that cleavage of PI3K proteins resulted in a decrease in lipid kinase activity of approximately 50% (Fig. 3B), indicating that cleavage of p110α by calpains results in a decrease of its lipid kinase activity.

We then investigated the effects of endogenous calpain activity on the stability of PI3K proteins in vivo. Calpain activity in serum-starved NIH 3T3 cells was inhibited with siRNA for CAPNS1 for 48 h (Fig. S1). WB analysis revealed that p110α levels were significantly increased by approximately 50% in cells with reduced calpain activity compared to control cells, whereas p110β levels were unaffected and p85 levels were slightly decreased (Fig. 3C). Similar results were obtained after treatment with the calpain inhibitor N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) for 48 h (Fig. S2), however, under these conditions p85 levels remained unchanged. These results are consistent with the notion that calpains have a role in regulating p110α levels in vivo.

Calpain Negatively Regulates PI3K/Akt Signaling.

In order to evaluate the effects of calpain activity on the modulation of PI3K/Akt signaling, NIH 3T3 cells were serum starved for 18 h and then treated with ALLN for 1 h prior to stimulation with 10% FBS for different lengths of time. The kinetics of serum-induced Akt phosphorylation (Fig. 4A) were found to be faster in cells treated with ALLN relative to DMSO-treated control cells. In addition, cells treated with ALLN showed a larger magnitude of serum-induced Akt phosphorylation relative to cells treated with vehicle (Fig. 4A). WB analysis of S6K phosphorylation demonstrated that the increased PI3K/Akt signaling activity was effectively propagated to downstream effectors of the pathway (Fig. 4B).

Fig. 4.

Effects of calpain activity on PI3K/Akt signaling. (A) Kinetics of Akt phosphorylation in response to 10% FBS stimulation in cells treated with ALLN or DMSO control. (B) Cells were treated as in A and analyzed for phosphorylation of S6K. (C) Akt phosphorylation in NIH 3T3 cells treated with ALLN and/or LY294002 and serum starved for 18 h, followed by stimulation with 10% FBS as indicated. (D) Akt phosphorylation, S6 phosphorylation, p110α expression, and calpain 2 expression in NIH 3T3 cells transfected with scrambled or CAPNS1 siRNA (50 nM) followed by 18 h of serum starvation and stimulation with 10% FBS as indicated 48 h after transfection. One representative experiment of three is shown for all blots.

Further experiments were performed to investigate the interplay between calpain activity and PI3K/Akt signaling. NIH 3T3 cells were serum starved for 18 h and treated for 1 h with ALLN and/or LY294002 (a dual PI3K/mTOR inhibitor), prior to stimulation with 10% FBS for 5 min as indicated. Consistent with previous data, treatment of cells with ALLN resulted in an increase of Akt phosphorylation at both Ser473 and Thr308 relative to vehicle-treated cells and higher levels of basal Akt phosphorylation were also observed (Fig. 4C). As expected, LY294002 treatment decreased Akt phosphorylation on both Akt sites (Fig. 4C). Interestingly, the observed increases in Akt phosphorylation upon calpain inhibition were not detected when cells were also treated with LY294002 (Fig. 4C). These results show that increased phosphorylation of Akt as a result of decreased calpain activity (Fig. 4 A and C) is dependent on LY294002 targets.

We also tested the impact of siRNA knockdown of CAPNS1 on PI3K pathway activity. NIH 3T3 cells were transfected with scrambled or CAPNS1 siRNA and 48 h later subjected to 18 h of serum starvation. Cells were then stimulated with 10% FBS followed by WB analysis (Fig. 4D). The expression of the catalytic subunit calpain 2, which is consistent with the down-regulation in expression of the regulatory subunit CAPNS1 (33), was significantly reduced by siRNA knockdown of CAPNS1. In line with the pharmacological data (Fig. 4A), Akt phosphorylation on Ser473 and Thr308 and S6 on Ser240/244 were enhanced in cells with reduced calpain expression (Fig. 4D). Furthermore, as seen in Fig. 3C, p110α levels were shown to be increased upon calpain inhibition with CAPNS1 siRNA. Taken together, these results reinforce the notion that calpains negatively modulate PI3K/Akt signaling activity in vivo.

Calpain Inhibition Increases Cell Viability During Serum Starvation.

We next investigated the functional impact of calpain activity on the PI3K/Akt pathway. NIH 3T3 cells were serum starved for 48 h, during which calpain and PI3K/mTOR activities were inhibited using pharmacological inhibitors or siRNA knockdown. Inhibition of calpain by ALLN resulted in an approximately twofold increase in the number of viable cells (Fig. 5A), whereas siRNA treatment increased cell numbers by approximately 50% (Fig. 5B). As expected, treatment with LY294002 reduced cell viability (Fig. 5 A and B). Interestingly, inhibition of calpain activity, with either ALLN or with CAPNS1 siRNA, in conjunction with LY294002 treatment opposed the effects of calpain inhibition in increasing viability (Fig. 5 A and B). These data thus suggest that the effects of calpain in modulating proliferative signals are at least partly dependent on PI3K/mTOR. These results also indicate that calpain has a role in the negative regulation of PI3K/mTOR-dependent growth and survival during serum starvation.

Fig. 5.

Effects of calpain activity on cell growth and survival. Viability of NIH 3T3 cells serum starved for 48 h and treated simultaneously with (A) DMSO, ALLN, and/or LY294002, or (B) scrambled siRNA, CAPNS1 siRNA, and/or LY294002. Data points are mean ± SEM (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Discussion

The activity of PI3Ks has to be tightly regulated to allow cells to grow and survive in a controlled fashion and to maintain tissue homeostasis (2, 35, 36). Accordingly, several mechanisms to control PI3K activity have evolved, some of which (highlighted in the introduction) are relatively well characterized (9, 10, 15, 19, 21, 37). Regulation by protein–protein interactions is a recurrent theme in biochemistry (12, 13) and several signaling pathway components are known to be regulated by the formation of protein complexes (14 –17). However, the extent by which protein interactors regulate class IA PI3Ks is not completely understood, perhaps because of the lack of off-the-shelf analytical tools to study transient and low-affinity binding partners.

It may be argued that, in order to regulate PI3K activity, functional binding partners would bind PI3K subunits with distinct stoichiometry as a function of PI3K activation status. Therefore, strategies commonly used to identify binding partners based on the overexpression of the protein bait may not always identify functional binders because functional interactions may on occasion occur through modifications or conformations not known a priori and not necessarily present in overexpressed proteins. We designed an AP-MS strategy based on quantitative LC-MS, which is emerging as a powerful method for the identification of protein–protein interactions (26, 38 –40) to identify functional binding partners of endogenous class IA PI3K. The reasoning was that regulatory proteins would be present at different levels in PI3K IPs extracted from stimulated and resting cells. Therefore, the use of quantitative MS was crucial for the success of these experiments as qualitative analyses did not identify any protein exclusively present in a set of PI3K IPs.

Quantitative MS identified three proteins that matched our (arbitrarily chosen) criteria to be considered a dynamic binding partner of PI3K (Fig. 1B). The selection criteria included proteins with a relatively modest fold change (twofold), as we anticipated a dynamic relationship, but with stringent MS data quality (both qualitative and quantitative) and biological reproducibility. CAPNS1 was consistently found to be more abundant in IPs taken from starved cells in all three experiments (Fig. 1E). CAPNS1 was not the candidate with the highest fold change; however, it was of immediate interest due to its known role in the cleavage of other signaling proteins (29) and its implication in Akt signaling (41 –43). Calpains have also been implicated in the regulation of multiple biological processes, including apoptosis, autophagy, proliferation, and migration (28, 29, 31).

Because of the lack of high-affinity antibodies against calpains, studies investigating this enzyme have typically used enzymatic assays to, for example, verify the genetic knockdown of CAPNS1 (32, 33). Using this analytical strategy, we demonstrated that the active calpain heterodimer associated with PI3K (Fig. 2B). Our results also suggest that the association between calpain and PI3K is enhanced in response to serum starvation, prompting us to speculate that the association may be an adaptive response to cellular stress.

Consistent with the function of calpains as proteases, p110α, p110β, and p85s (the class IA PI3K subunits expressed in NIH 3T3 cells; ref. 19) were in vitro substrates of calpain 1 and calpain 2 (Fig. 3A). However, we observed that p110β was cleaved to a much lower extent than p110α, demonstrating that calpain preferentially cleaves p110α over p110β in this assay. Subsequent experiments also indicated that calpain has a role in down-regulating PI3K activity in vitro (Fig. 3B). Furthermore, inhibition of calpain activity resulted in increased levels of p110α in vivo, whereas p85 and p110β levels were found to be minimally affected by calpain inhibition (Fig. 3C and Fig. S2).

The regulation of PI3K activity and expression by calpains resulted in a modulation of downstream signaling events. Indeed, pharmacological and siRNA inhibition of both calpain isoforms produced an enhancement of PI3K pathway activity as assessed by phosphorylation of downstream proteins (Fig. 4 A, B, and D) and cell viability during serum starvation (Fig. 5). Because these activities appear to be dependent on PI3K/mTOR (Fig. 4C and Fig. 5), we propose that calpain negatively regulates the PI3K/Akt signaling pathway activity via its interaction with PI3K. Pharmacological inhibition of calpain was previously shown to induce Akt phosphorylation in human neutrophils and monocytes, although no mechanism was described (42, 43). However, another study showed that CAPNS1 knockout in mouse embryonic fibroblasts resulted in decreased Akt phosphorylation compared to wild-type cells when treated acutely with media free from amino acids and serum, and it was proposed that the phosphatase PP2A is a substrate of calpain (41). Our results indicate that the association between PI3K and calpain increases as serum starvation is prolonged; therefore, the PI3K-mediated effects of calpain on Akt phosphorylation that we observed are unlikely to occur during the short starvation treatments (up to 20 min) used by Bertoli et al (41). Taken together, these data reflect the complexity of the regulation of cell signaling in vivo and suggest that calpains may have a role in the regulation of multiple nodes of the signaling network.

Recently, it has been proposed that cellular PI3K levels are dynamic and that this confers protection to the overall cell population in a culture because the PI3K/Akt pathway is only active in a subset of cells at a given time (44). This work by Yuan et al. showed that p110α levels in immortalized human MCF10A breast epithelial cells were regulated through a cycle of proteasomal degradation and resynthesis. Here we have shown that calpain protease activity may have a role in this process, or it may represent an additional mechanism for the regulation of p110α stability under certain circumstances, such as during stress.

In conclusion, our results demonstrate that calpains interact with class IA PI3K and that this interaction has a functional role in controlling PI3K p110α activity and stability. Our work further suggests that this calpain–PI3K interaction may have an important role in the regulation of signaling activity in response to stress during serum starvation, and thus it could potentially be a previously undiscovered mechanism by which PI3K/Akt pathway deregulation contributes to pathogenesis.

Materials and Methods

Antibodies and Reagents.

See SI Materials and Methods for further details.

Cell Culture.

NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO₂ atmosphere.

Immunoprecipitation.

Cells were lysed in buffer containing 50 mM Tris·HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.3% CHAPS. Clarified lysates were precleared with sepharose G prior to incubation with antibodies for 2–18 h at 4 °C followed by incubation for 1.5 h with sepharose G at 4 °C. For p85 IPs a 50∶50 mixture of anti-p85 06–195 (Upstate) and anti-p85 06–497 (Upstate) was used at a ratio of 2 μL per milligram protein. For calpain IPs a 50∶50 mixture of anti-calpain sc-30064 (Santa Cruz Biotechology) and anti-calpain 2 2539 (Cell Signaling Technology) was used at a ratio of 20 μL per milligram protein.

In-Gel Digestion.

Purified protein complexes were loaded on a 1D 10% SDS-PAGE. After separation, proteins were visualized by colloidal Coomassie brilliant blue staining and the gel was sectioned. Digestion of gel-immobilized proteins was performed as described in ref. 45 except that extracted peptides were dried using a Christ Rotational Vacuum Concentrator (RVC 2–25) before resuspension in 0.1% (vol/vol) trifluoracetic acid.

LC-MS/MS.

LC-MS/MS analysis was performed as described in ref. 46 (SI Materials and Methods).

MS Data Analysis.

Mascot search engine (v2.2.02) was used for protein identification (47) and Pescal was used for protein quantification (48) (SI Materials and Methods).

Lipid Kinase Assay.

Lipid kinase assay for PI3K activity was performed as described in ref. 34.

Immunoblotting.

Cells were lysed in buffer containing 1% Triton X-100 and denatured by boiling with DTT-containing loading buffer. Proteins were resolved using 8–12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes, prior to blocking with 5% milk and incubation with primary antibodies. Binding was visualized using horseradish peroxidise-conjugated secondary antibodies and luminescence.

Acknowledgments

We thank the members of the Center for Cell Signaling, in particular Khaled Ali and Maria Whitehead, for helpful discussions and feedback. The study was funded by grants from the Medical Research Council (L.B. PhD Studentship), Barts and the London Charity (297/997), and the National Institute for Health Research. C.C. was supported by an European Union Marie Curie Fellowship (PIIF-GA-2009-252846).

Footnotes

↵¹To whom correspondence should be addressed. E-mail: p.cutillas{at}qmul.ac.uk.

Author contributions: P.R.C. designed research; L.B. and C.C. performed research; L.B., C.C., B.V., and P.R.C. analyzed data; and L.B. and P.R.C. wrote the paper.
The authors declare a conflict of interest. P.R.C. and B.V. are founders and consultants of Activiomics, Ltd. B.V. is also a consultant to Intellikine and GlaxoSmithKline.
This article is a PNAS Direct Submission. J.R.H. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1107692108/-/DCSupplemental.

Freely available online through the PNAS open access option.

References

↵
1. Cantley LC
(2002) The phosphoinositide 3-kinase pathway. Science 296:1655–1657.
OpenUrl Abstract/FREE Full Text
↵
1. Manning BD,
2. Cantley LC
(2007) AKT/PKB signaling: Navigating downstream. Cell 129:1261–1274.
OpenUrl CrossRef PubMed
↵
1. Vanhaesebroeck B,
2. Guillermet-Guibert J,
3. Graupera M,
4. Bilanges B
(2010) The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol 11:329–341.
OpenUrl CrossRef PubMed
↵
1. Terauchi Y,
2. et al.
(1999) Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase. Nat Genet 21:230–235.
OpenUrl CrossRef PubMed
↵
1. Randis TM,
2. Puri KD,
3. Zhou H,
4. Diacovo TG
(2008) Role of PI3Kδ and PI3Kγ in inflammatory arthritis and tissue localization of neutrophils. Eur J Immunol 38:1215–1224.
OpenUrl CrossRef PubMed
↵
1. Ohta H,
2. Arai S,
3. Akita K,
4. Ohta T,
5. Fukuda S
(2011) Neurotrophic effects of a cyanine dye via the PI3K-Akt pathway: Attenuation of motor discoordination and neurodegeneration in an ataxic animal model. PLoS One 6:e17137.
OpenUrl CrossRef PubMed
↵
1. Samuels Y,
2. Velculescu VE
(2004) Oncogenic mutations of PIK3CA in human cancers. Cell Cycle 3:1221–1224.
OpenUrl PubMed
↵
1. Samuels Y,
2. et al.
(2005) Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7:561–573.
OpenUrl CrossRef PubMed
↵
1. Myers MP,
2. et al.
(1998) The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc Natl Acad Sci USA 95:13513–13518.
OpenUrl Abstract/FREE Full Text
↵
1. Resjo S,
2. et al.
(2002) Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cell Signal 14:231–238.
OpenUrl CrossRef PubMed
↵
1. O'Reilly KE,
2. et al.
(2006) mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66:1500–1508.
OpenUrl Abstract/FREE Full Text
↵
1. Jones S,
2. Thornton JM
(1996) Principles of protein–protein interactions. Proc Natl Acad Sci USA 93:13–20.
OpenUrl Abstract/FREE Full Text
↵
1. Uetz P,
2. et al.
(2000) A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623–627.
OpenUrl CrossRef PubMed
↵
1. von Kriegsheim A,
2. et al.
(2009) Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 11:1458–1464.
OpenUrl CrossRef PubMed
↵
1. Brazil DP,
2. Park J,
3. Hemmings BA
(2002) PKB binding proteins: Getting in on the Akt. Cell 111:293–303.
OpenUrl CrossRef PubMed
↵
1. Kim D-H,
2. et al.
(2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175.
OpenUrl CrossRef PubMed
↵
1. Sarbassov DD,
2. et al.
(2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14:1296–1302.
OpenUrl CrossRef PubMed
↵
1. Yu J,
2. et al.
(1998) Regulation of the p85/p110 phosphatidylinositol 3′-kinase: Stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol 18:1379–1387.
OpenUrl Abstract/FREE Full Text
↵
1. Geering B,
2. Cutillas PR,
3. Nock G,
4. Gharbi SI,
5. Vanhaesebroeck B
(2007) Class IA phosphoinositide 3-kinases are obligate p85–p110 heterodimers. Proc Natl Acad Sci USA 104:7809–7814.
OpenUrl Abstract/FREE Full Text
↵
1. Kodaki T,
2. Woscholski R,
3. Hallberg B,
4. Rodriguez-Viciana Julian Downward P,
5. Parker PJ
(1994) The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol 4:798–806.
OpenUrl CrossRef PubMed
↵
1. Gupta S,
2. et al.
(2007) Binding of Ras to phosphoinositide 3-kinase p110a is required for Ras-driven Tumorigenesis in mice. Cell 129:957–968.
OpenUrl CrossRef PubMed
↵
1. Gupta N,
2. et al.
(1999) The SH2 domain-containing inositol 5′-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRllb1-mediated inhibition of B cell receptor signaling. J Biol Chem 274:7489–7494.
OpenUrl Abstract/FREE Full Text
↵
1. Perez OD,
2. et al.
(2002) Activation of the PKB/AKT pathway by ICAM-2. Immunity 16:51–65.
OpenUrl CrossRef PubMed
↵
1. Vogel LB,
2. Fujita DJ
(1993) The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3′-kinase from T lymphocytes. Mol Cell Biol 13:7408–7417.
OpenUrl Abstract/FREE Full Text
↵
1. Rabinovsky R,
2. et al.
(2009) p85 associates with unphosphorylated PTEN and the PTEN-associated complex. Mol Cell Biol 29:5377–5388.
OpenUrl Abstract/FREE Full Text
↵
1. Dengjel J,
2. Kratchmarova I,
3. Blagoev B
(2010) Mapping protein–protein interactions by quantitative proteomics. Methods Mol Biol 658:267–278.
OpenUrl CrossRef PubMed
↵
1. Goll DE,
2. Thompson VF,
3. Li H,
4. Wei W,
5. Cong J
(2003) The calpain system. Physiol Rev 83:731–801.
OpenUrl Abstract/FREE Full Text
↵
1. Perrin BJ,
2. Huttenlocher A
(2002) Calpain. Int J Biochem Cell Biol 34:722–725.
OpenUrl CrossRef PubMed
↵
1. Franco SJ,
2. Huttenlocher A
(2005) Regulating cell migration: Calpains make the cut. J Cell Sci 118:3829–3838.
OpenUrl Abstract/FREE Full Text
↵
1. Carragher NO,
2. Frame MC
(2002) Calpain: A role in cell transformation and migration. Int J Biochem Cell Biol 34:1539–1543.
OpenUrl CrossRef PubMed
↵
1. Demarchi F,
2. Schneider C
(2007) The Calpain system as a modulator of stress/damage response. Cell Cycle 6:136–138.
OpenUrl PubMed
↵
1. Dourdin N,
2. et al.
(2001) Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts. J Biol Chem 276:48382–48388.
OpenUrl Abstract/FREE Full Text
↵
1. Tan Y,
2. Wu C,
3. De Veyra T,
4. Greer PA
(2006) Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli. J Biol Chem 281:17689–17698.
OpenUrl Abstract/FREE Full Text
↵
1. Chaussade C,
2. et al.
(2007) Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem J 404:449–458.
OpenUrl CrossRef PubMed
↵
1. Leevers SJ,
2. Vanhaesebroeck B,
3. Waterfield MD
(1999) Signalling through phosphoinositide 3-kinases: The lipids take centre stage. Curr Opin Cell Biol 11:219–225.
OpenUrl CrossRef PubMed
↵
1. Engelman JA,
2. Luo J,
3. Cantley LC
(2006) The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 7:606–619.
OpenUrl CrossRef PubMed
↵
1. Kok K,
2. Geering B,
3. Vanhaesebroeck B
(2009) Regulation of phosphoinositide 3-kinase expression in health and disease. Trends Biochem Sci 34:115–127.
OpenUrl CrossRef PubMed
↵
1. Gavin AC,
2. et al.
(2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440:631–636.
OpenUrl CrossRef PubMed
↵
1. Guerrero C,
2. Tagwerker C,
3. Kaiser P,
4. Huang L
(2006) An integrated mass spectrometry-based proteomic approach: Quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (qtax) to decipher the 26 s proteasome-interacting network. Mol Cell Proteomics 5:366–378.
OpenUrl Abstract/FREE Full Text
↵
1. Trinkle-Mulcahy L,
2. et al.
(2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol 183:223–239.
OpenUrl Abstract/FREE Full Text
↵
1. Bertoli C,
2. Copetti T,
3. Lam EWF,
4. Demarchi F,
5. Schneider C
(2008) Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A. Oncogene 28:721–733.
OpenUrl PubMed
↵
1. Katsube M,
2. et al.
(2008) Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils. J Leukoc Biol 84:255–263.
OpenUrl Abstract/FREE Full Text
↵
1. Noma H,
2. et al.
(2009) Calpain inhibition induces activation of the distinct signalling pathways and cell migration in human monocytes. Immunology 128:e487–496.
OpenUrl CrossRef PubMed
↵
1. Yuan TL,
2. Wulf G,
3. Burga L,
4. Cantley LC
(2011) Cell-to-cell variability in PI3K protein level regulates PI3K-AKT pathway activity in cell populations. Curr Biol 21:173–183.
OpenUrl CrossRef PubMed
↵
1. Cutillas PR,
2. Geering B,
3. Waterfield MD,
4. Vanhaesebroeck B
(2005) Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: Analysis of phosphoprotein dynamics in a B cell lymphoma cell line. Mol Cell Proteomics 4:1038–1051.
OpenUrl Abstract/FREE Full Text
↵
1. Casado P,
2. Cutillas PR
(2011) A self-validating quantitative mass spectrometry method for assessing the accuracy of high-content phosphoproteomic experiments. Mol Cell Proteomics 10:10.1074/mcp.M110.003079.
↵
1. Perkins DN,
2. Pappin DJC,
3. Creasy DM,
4. Cottrell JS
(1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567.
OpenUrl CrossRef PubMed
↵
1. Cutillas PR,
2. Vanhaesebroeck B
(2007) Quantitative profile of five murine core proteomes using label-free functional proteomics. Mol Cell Proteomics 6:1560–1573.
OpenUrl Abstract/FREE Full Text

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Biological Sciences
Biochemistry

Proceedings of the National Academy of Sciences: 108 (39)

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[1] ↵
Cantley LC
(2002) The phosphoinositide 3-kinase pathway. Science 296:1655–1657.
OpenUrl Abstract/FREE Full Text

[2] Cantley LC

[3] ↵
Manning BD,
Cantley LC
(2007) AKT/PKB signaling: Navigating downstream. Cell 129:1261–1274.
OpenUrl CrossRef PubMed

[4] Manning BD,

[5] Cantley LC

[6] ↵
Vanhaesebroeck B,
Guillermet-Guibert J,
Graupera M,
Bilanges B
(2010) The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol 11:329–341.
OpenUrl CrossRef PubMed

[7] Vanhaesebroeck B,

[8] Guillermet-Guibert J,

[9] Graupera M,

[10] Bilanges B

[11] ↵
Terauchi Y,
et al.
(1999) Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase. Nat Genet 21:230–235.
OpenUrl CrossRef PubMed

[12] Terauchi Y,

[13] et al.

[14] ↵
Randis TM,
Puri KD,
Zhou H,
Diacovo TG
(2008) Role of PI3Kδ and PI3Kγ in inflammatory arthritis and tissue localization of neutrophils. Eur J Immunol 38:1215–1224.
OpenUrl CrossRef PubMed

[15] Randis TM,

[16] Puri KD,

[17] Zhou H,

[18] Diacovo TG

[19] ↵
Ohta H,
Arai S,
Akita K,
Ohta T,
Fukuda S
(2011) Neurotrophic effects of a cyanine dye via the PI3K-Akt pathway: Attenuation of motor discoordination and neurodegeneration in an ataxic animal model. PLoS One 6:e17137.
OpenUrl CrossRef PubMed

[20] Ohta H,

[21] Arai S,

[22] Akita K,

[23] Ohta T,

[24] Fukuda S

[25] ↵
Samuels Y,
Velculescu VE
(2004) Oncogenic mutations of PIK3CA in human cancers. Cell Cycle 3:1221–1224.
OpenUrl PubMed

[26] Samuels Y,

[27] Velculescu VE

[28] ↵
Samuels Y,
et al.
(2005) Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7:561–573.
OpenUrl CrossRef PubMed

[29] Samuels Y,

[30] et al.

[31] ↵
Myers MP,
et al.
(1998) The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc Natl Acad Sci USA 95:13513–13518.
OpenUrl Abstract/FREE Full Text

[32] Myers MP,

[33] et al.

[34] ↵
Resjo S,
et al.
(2002) Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cell Signal 14:231–238.
OpenUrl CrossRef PubMed

[35] Resjo S,

[36] et al.

[37] ↵
O'Reilly KE,
et al.
(2006) mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66:1500–1508.
OpenUrl Abstract/FREE Full Text

[38] O'Reilly KE,

[39] et al.

[40] ↵
Jones S,
Thornton JM
(1996) Principles of protein–protein interactions. Proc Natl Acad Sci USA 93:13–20.
OpenUrl Abstract/FREE Full Text

[41] Jones S,

[42] Thornton JM

[43] ↵
Uetz P,
et al.
(2000) A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623–627.
OpenUrl CrossRef PubMed

[44] Uetz P,

[45] et al.

[46] ↵
von Kriegsheim A,
et al.
(2009) Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 11:1458–1464.
OpenUrl CrossRef PubMed

[47] von Kriegsheim A,

[48] et al.

[49] ↵
Brazil DP,
Park J,
Hemmings BA
(2002) PKB binding proteins: Getting in on the Akt. Cell 111:293–303.
OpenUrl CrossRef PubMed

[50] Brazil DP,

[51] Park J,

[52] Hemmings BA

[53] ↵
Kim D-H,
et al.
(2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175.
OpenUrl CrossRef PubMed

[54] Kim D-H,

[55] et al.

[56] ↵
Sarbassov DD,
et al.
(2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14:1296–1302.
OpenUrl CrossRef PubMed

[57] Sarbassov DD,

[58] et al.

[59] ↵
Yu J,
et al.
(1998) Regulation of the p85/p110 phosphatidylinositol 3′-kinase: Stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol 18:1379–1387.
OpenUrl Abstract/FREE Full Text

[60] Yu J,

[61] et al.

[62] ↵
Geering B,
Cutillas PR,
Nock G,
Gharbi SI,
Vanhaesebroeck B
(2007) Class IA phosphoinositide 3-kinases are obligate p85–p110 heterodimers. Proc Natl Acad Sci USA 104:7809–7814.
OpenUrl Abstract/FREE Full Text

[63] Geering B,

[64] Cutillas PR,

[65] Nock G,

[66] Gharbi SI,

[67] Vanhaesebroeck B

[68] ↵
Kodaki T,
Woscholski R,
Hallberg B,
Rodriguez-Viciana Julian Downward P,
Parker PJ
(1994) The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol 4:798–806.
OpenUrl CrossRef PubMed

[69] Kodaki T,

[70] Woscholski R,

[71] Hallberg B,

[72] Rodriguez-Viciana Julian Downward P,

[73] Parker PJ

[74] ↵
Gupta S,
et al.
(2007) Binding of Ras to phosphoinositide 3-kinase p110a is required for Ras-driven Tumorigenesis in mice. Cell 129:957–968.
OpenUrl CrossRef PubMed

[75] Gupta S,

[76] et al.

[77] ↵
Gupta N,
et al.
(1999) The SH2 domain-containing inositol 5′-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRllb1-mediated inhibition of B cell receptor signaling. J Biol Chem 274:7489–7494.
OpenUrl Abstract/FREE Full Text

[78] Gupta N,

[79] et al.

[80] ↵
Perez OD,
et al.
(2002) Activation of the PKB/AKT pathway by ICAM-2. Immunity 16:51–65.
OpenUrl CrossRef PubMed

[81] Perez OD,

[82] et al.

[83] ↵
Vogel LB,
Fujita DJ
(1993) The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3′-kinase from T lymphocytes. Mol Cell Biol 13:7408–7417.
OpenUrl Abstract/FREE Full Text

[84] Vogel LB,

[85] Fujita DJ

[86] ↵
Rabinovsky R,
et al.
(2009) p85 associates with unphosphorylated PTEN and the PTEN-associated complex. Mol Cell Biol 29:5377–5388.
OpenUrl Abstract/FREE Full Text

[87] Rabinovsky R,

[88] et al.

[89] ↵
Dengjel J,
Kratchmarova I,
Blagoev B
(2010) Mapping protein–protein interactions by quantitative proteomics. Methods Mol Biol 658:267–278.
OpenUrl CrossRef PubMed

[90] Dengjel J,

[91] Kratchmarova I,

[92] Blagoev B

[93] ↵
Goll DE,
Thompson VF,
Li H,
Wei W,
Cong J
(2003) The calpain system. Physiol Rev 83:731–801.
OpenUrl Abstract/FREE Full Text

[94] Goll DE,

[95] Thompson VF,

[96] Li H,

[97] Wei W,

[98] Cong J

[99] ↵
Perrin BJ,
Huttenlocher A
(2002) Calpain. Int J Biochem Cell Biol 34:722–725.
OpenUrl CrossRef PubMed

[100] Perrin BJ,

[101] Huttenlocher A

[102] ↵
Franco SJ,
Huttenlocher A
(2005) Regulating cell migration: Calpains make the cut. J Cell Sci 118:3829–3838.
OpenUrl Abstract/FREE Full Text

[103] Franco SJ,

[104] Huttenlocher A

[105] ↵
Carragher NO,
Frame MC
(2002) Calpain: A role in cell transformation and migration. Int J Biochem Cell Biol 34:1539–1543.
OpenUrl CrossRef PubMed

[106] Carragher NO,

[107] Frame MC

[108] ↵
Demarchi F,
Schneider C
(2007) The Calpain system as a modulator of stress/damage response. Cell Cycle 6:136–138.
OpenUrl PubMed

[109] Demarchi F,

[110] Schneider C

[111] ↵
Dourdin N,
et al.
(2001) Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts. J Biol Chem 276:48382–48388.
OpenUrl Abstract/FREE Full Text

[112] Dourdin N,

[113] et al.

[114] ↵
Tan Y,
Wu C,
De Veyra T,
Greer PA
(2006) Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli. J Biol Chem 281:17689–17698.
OpenUrl Abstract/FREE Full Text

[115] Tan Y,

[116] Wu C,

[117] De Veyra T,

[118] Greer PA

[119] ↵
Chaussade C,
et al.
(2007) Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem J 404:449–458.
OpenUrl CrossRef PubMed

[120] Chaussade C,

[121] et al.

[122] ↵
Leevers SJ,
Vanhaesebroeck B,
Waterfield MD
(1999) Signalling through phosphoinositide 3-kinases: The lipids take centre stage. Curr Opin Cell Biol 11:219–225.
OpenUrl CrossRef PubMed

[123] Leevers SJ,

[124] Vanhaesebroeck B,

[125] Waterfield MD

[126] ↵
Engelman JA,
Luo J,
Cantley LC
(2006) The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 7:606–619.
OpenUrl CrossRef PubMed

[127] Engelman JA,

[128] Luo J,

[129] Cantley LC

[130] ↵
Kok K,
Geering B,
Vanhaesebroeck B
(2009) Regulation of phosphoinositide 3-kinase expression in health and disease. Trends Biochem Sci 34:115–127.
OpenUrl CrossRef PubMed

[131] Kok K,

[132] Geering B,

[133] Vanhaesebroeck B

[134] ↵
Gavin AC,
et al.
(2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440:631–636.
OpenUrl CrossRef PubMed

[135] Gavin AC,

[136] et al.

[137] ↵
Guerrero C,
Tagwerker C,
Kaiser P,
Huang L
(2006) An integrated mass spectrometry-based proteomic approach: Quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (qtax) to decipher the 26 s proteasome-interacting network. Mol Cell Proteomics 5:366–378.
OpenUrl Abstract/FREE Full Text

[138] Guerrero C,

[139] Tagwerker C,

[140] Kaiser P,

[141] Huang L

[142] ↵
Trinkle-Mulcahy L,
et al.
(2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol 183:223–239.
OpenUrl Abstract/FREE Full Text

[143] Trinkle-Mulcahy L,

[144] et al.

[145] ↵
Bertoli C,
Copetti T,
Lam EWF,
Demarchi F,
Schneider C
(2008) Calpain small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A. Oncogene 28:721–733.
OpenUrl PubMed

[146] Bertoli C,

[147] Copetti T,

[148] Lam EWF,

[149] Demarchi F,

[150] Schneider C

[151] ↵
Katsube M,
et al.
(2008) Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils. J Leukoc Biol 84:255–263.
OpenUrl Abstract/FREE Full Text

[152] Katsube M,

[153] et al.

[154] ↵
Noma H,
et al.
(2009) Calpain inhibition induces activation of the distinct signalling pathways and cell migration in human monocytes. Immunology 128:e487–496.
OpenUrl CrossRef PubMed

[155] Noma H,

[156] et al.

[157] ↵
Yuan TL,
Wulf G,
Burga L,
Cantley LC
(2011) Cell-to-cell variability in PI3K protein level regulates PI3K-AKT pathway activity in cell populations. Curr Biol 21:173–183.
OpenUrl CrossRef PubMed

[158] Yuan TL,

[159] Wulf G,

[160] Burga L,

[161] Cantley LC

[162] ↵
Cutillas PR,
Geering B,
Waterfield MD,
Vanhaesebroeck B
(2005) Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: Analysis of phosphoprotein dynamics in a B cell lymphoma cell line. Mol Cell Proteomics 4:1038–1051.
OpenUrl Abstract/FREE Full Text

[163] Cutillas PR,

[164] Geering B,

[165] Waterfield MD,

[166] Vanhaesebroeck B

[167] ↵
Casado P,
Cutillas PR
(2011) A self-validating quantitative mass spectrometry method for assessing the accuracy of high-content phosphoproteomic experiments. Mol Cell Proteomics 10:10.1074/mcp.M110.003079.

[168] Casado P,

[169] Cutillas PR

[170] ↵
Perkins DN,
Pappin DJC,
Creasy DM,
Cottrell JS
(1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567.
OpenUrl CrossRef PubMed

[171] Perkins DN,

[172] Pappin DJC,

[173] Creasy DM,

[174] Cottrell JS

[175] ↵
Cutillas PR,
Vanhaesebroeck B
(2007) Quantitative profile of five murine core proteomes using label-free functional proteomics. Mol Cell Proteomics 6:1560–1573.
OpenUrl Abstract/FREE Full Text

[176] Cutillas PR,

[177] Vanhaesebroeck B

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Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Abstract

Results

AP-MS Study Suggests That CAPNS1 Interacts with PI3K in NIH 3T3 Cells as a Function of Pathway Activation.

Calpain and PI3K Interact Dynamically.

Calpain 1 and 2 Cleave p110α and p85 and Reduce PI3K Enzymatic Activity.

Calpain Negatively Regulates PI3K/Akt Signaling.

Calpain Inhibition Increases Cell Viability During Serum Starvation.

Discussion

Materials and Methods

Antibodies and Reagents.

Cell Culture.

Immunoprecipitation.

In-Gel Digestion.

LC-MS/MS.

MS Data Analysis.

Lipid Kinase Assay.

Immunoblotting.

Acknowledgments

Footnotes

References

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Abstract

Results

AP-MS Study Suggests That CAPNS1 Interacts with PI3K in NIH 3T3 Cells as a Function of Pathway Activation.

Calpain and PI3K Interact Dynamically.

Calpain 1 and 2 Cleave p110α and p85 and Reduce PI3K Enzymatic Activity.

Calpain Negatively Regulates PI3K/Akt Signaling.

Calpain Inhibition Increases Cell Viability During Serum Starvation.

Discussion

Materials and Methods

Antibodies and Reagents.

Cell Culture.

Immunoprecipitation.

In-Gel Digestion.

LC-MS/MS.

MS Data Analysis.

Lipid Kinase Assay.

Immunoblotting.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

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