Significance of filamin A in mTORC2 function in glioblastoma

Abstract

Background: Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes.

Methods: To further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed.

Results: In addition to the inhibition of mTORC2 activity, we demonstrated significant alteration of actin distribution as revealed by the use of phalloidin staining. Furthermore, vinculin staining was altered which suggests changes in focal adhesion. Inhibition of cell migration and invasion was observed with PP242. Two major proteins that are associated with this mTORC2 multiprotein complex were found. Mass spectrometry identified one of them as Filamin A (FLNA). Association of FLNA with RICTOR but not mTOR was demonstrated. Moreover, in vitro, purified mTORC2 can phosphorylate FLNA likewise its known substrate, AKT. In GBM cells, colocalization of FLNA with RICTOR was observed, and the overall amounts of FLNA protein as well as phosphorylated FLNA are high. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decreased. This treatment also disrupted colocalization of Actin filaments and FLNA.

Conclusions: Our results support FLNA as a new downstream effector of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA signaling pathway plays important roles in motility and invasion of glioblastoma cells.

Fig. 1

PP242 inhibits mTORC2 and exerts significant effects on actin cytoskeleton and focal adhesion of U87vIII cells. a PP242 inhibits mTORC1 and mTORC2 activities effectively while rapamycin only inhibits mTORC1 activity. Cells were treated with inhibitors for 24 h and phosphorylation of AKT and S6 was examined as described in Methods. b U87vIII cells have constitutively active mTORC1 and mTORC2 activities in starved condition. Stimulation by amino acids and serum fully activate mTOR kinase activity. A 3-h treatment of 2.5 μM PP242 ablates phosphorylation of AKT and S6. c Actin cytoskeleton and focal adhesion of U87vIII cells treated with PP242 for 24 h were examined using immunofluorescence staining by Phalloidin (actin) and anti-vinculin (focal adhesion) (Exposure time: actin 254 ms; vinculin 251 ms). Effects of PP242 on these processes were examined by treating cells with PP242. Effects on actin cytoskeleton rearrangement are clearly visible on the thickness of actin layers on surrounding cell membrane (arrows)

Fig. 2

mTOR inhibition affects cell migration and invasion. a Wound-healing migration assay of U87vIII cells grown in a single layer. Space between a scratch is approximately 1 mm wide. Pictures were taken at 12 h after the scratch was made for each sample. Migration of cells treated with PP242, rapamycin, or U0126 was examined. b Numbers of invaded U87vIII cells through modified Boyden chambers were determined as described in Methods. Effects of PP242 at 2.5 and 5.0 μM, rapamycin (100 nM) or U0126 (10.0 μM) are shown. Cells were incubated with the inhibitor for 6 h. Invaded cells attaching to the chamber membrane were stained and the number of cells for each sample group was determined by measuring the absorbance values of crystal violet-stained cells at 590 nm. (P2.5 = PP242 2.5 μM, P5.0 = PP242 5.0 μM, RAPA = rapamycin 100 nM, U10 = U0126 10.0 μM) Data are the mean ± SD (n = 3). *P < 0.05; **P < 0.005, P-values from two-tailed student t-test (unpaired comparison)

Fig. 3

Purification, identification, and characterization of mTORC2 and its binding partners. a Silver-stained high molecular weight mTORC2 components purified from U87vIII cells stably expressing FLAG-RICTOR are shown. Four large proteins (numbered as 1-4) were analyzed by mass spectrometry after being run on 7 % SDS-PAGE gel, excised, then digested by Trypsin. Mass spectrometry results are summarized in the table on the right. Filamin A (FLNA) and Myosin-9 (MYH9) represent unknown bands 1 and 3, respectively. †Mascot protein score; a protein with score value >21 with >2 unique peptides is considered significant (P <0.05). b Immunoblots of purified proteins from U87vIII cells showing main components of mTORC2 (mTOR, RICTOR, SIN1) including phosphorylated and total FLNA. c Immunoprecipitation of U87vIII cell lysate by protein G Dynabeads coupled with antibodies against main components of mTOR complex 1 and 2. Phosphorylated FLNA was co-immunoprecipitated with anti-mTOR and anti-RICTOR coated beads while these two components of mTORC2 were pulled down with anti-pFLNA coated beads. RAPTOR, an mTORC1-specific component, can be co-immunoprecipitated with anti-mTOR coated beads but not with anti-pFLNA beads

Fig. 4

RICTOR binds FLNA. a FLAG-RICTOR is pulled down after lysing U87vIII cells with CHAPS or Triton X-100 as a detergent in lysis buffer. An immunoblot shows that RICTOR is associated with mTOR and FLNA in samples from CHAPS-treated extracts but only with FLNA in Triton X-100-treated extracts b Immunofluoresence labeling of two individual U87vIII cells grown in normal condition. Majority of FLNA and RICTOR colocalize along cell membrane and cytoplasm. Anti-FLNA antibody and Anti-RICTOR antibody were used to stain FLNA and RICTOR, respectively (Exposure time: FLNA 334 ms; RICTOR 540 ms; DAPI 41 ms). However, only RICTOR is found in nuclei of the cells. Overall results indicate that FLNA is an mTORC2 associated protein which is a binding partner of RICTOR

Fig. 5

Purified mTORC2 phosphorylates FLNA at Ser2152 in vitro. a In vitro kinase assay of mTORC2 purified from U87vIII cells with FLNA and AKT as substrates. Level of phosphorylated FLNA (Ser2152) and phosphorylated AKT (Ser473) were increased in the presence of mTORC2 and ATP. Negative control groups contain either no purified complex or no ATP. Activity of mTORC2 is maximal when Mn²⁺ is used instead of Mg²⁺. b In vitro kinase assay of mTORC2 purified from U87vIII cells with FLNA as a substrate. Two concentrations of PP242 (+ = 1.25 μM; ++ = 5.0 μM) and one concentration of IPA3 (+ = 40 μM) were added into three samples to inhibit the kinase activity of mTORC2. Levels of pFLNA and pAKT when treated with PP242 are decreased compared to ones without PP242

Fig. 6

mTORC2 regulates FLNA phosphorylation at Ser2152. a An immunoblot showing high amounts of phosphorylated FLNA (pFLNA) and phosphorylated AKT (pAKT), a known mTORC2 substrate, in U87vIII cell lysate. In contrast, the amount of mTOR is similar in both U87vIII and HEK293T cells. b Level of FLNA phosphorylation (Ser2152) in U87vIII cells treated with two different concentrations of RICTOR siRNA decreases correlating to mTORC2 inhibition as detected by pAKT at Ser473. (Sc: scrambled siRNA, + = 4 μL, ++ = 8 μL siRNA). c Immunofluorescence staining of actin filaments, FLNA and RICTOR (Exposure time: RICTOR 318 ms; actin 580 ms; FLNA 123 ms). Results obtained from RICTOR knockdown experiment (48-h incubation with RICTOR siRNA) as described in Methods were used to observe effects of mTORC2 loss on arrangement of actin cytoskeleton and FLNA localization

Fig. 7

PP242 inhibits FLNA phosphorylation and causes dissociation of FLNA from actin cytoskeleton. a Levels of pFLNA (Ser2152) in U87vIII cells treated with different concentrations (0.31-5.0 μM) of PP242 were examined by carrying out Western analysis using anti-phospho-FLNA antibody. Average relative signal intensity of pFLNA adjusted by total FLNA amount of each condition is shown. Error bars indicate ± SD (n = 3). b Effects of PP242 on actin cytoskeleton and FLNA localization were examined by treating U87vIII cells with PP242 (2.5 μM or 5.0 μM) for 24 h before fixation. Immunofluorescence staining of actin filaments and FLNA was carried out as described in Methods (Exposure time: FLNA130 ms; actin 250 ms; DAPI 55 ms). Colocalization of Actin and FLNA is interrupted in samples treated with PP242. The results resemble effects of the treatment with siRNA against RICTOR