TWIST1 Heterodimerization with E12 Requires Coordinated Protein Phosphorylation to Regulate Periostin Expression

Figure 1. Identification and functional characterization of phosphorylation at S68 residue in TW. (A) Detection of TW S68 phosphorylation and total TW in patient GBM samples compared to normal brains. (B) TW S68 phosphorylation at the endogenous level in T98G cells detected by immunoprecipitation with pTW S68 antibody followed by detection using a total TW antibody. As a negative control non-specific same isotype IgG was used. Inputs were 2% of total proteins used for immunoprecipitation. (C) Expression of pTW S68 and total TW in primary glioma stem cells GBM4 and G131. (D) Relative fraction of pTW S68 in total amount of TW immunoprecipitated from U87MG (dTW-A) and GBM8 cells with TW overexpression. The percent of pTWS68 is defined as area under the curve of phospho-peptide divided by sum of pTWS68 + Non-phospho-peptide and averaged from three biological replicates. (E) Non-modified and phosphopeptides detected in U87MG (dTW-A) and GBM8 cells with TW overexpression and used for calculation of S68 phosphorylation fraction in panel D (see Figure S2). (F) Confirmation of comparable levels of TW and TW(S68A) mutant overexpression in T98G cells and corresponding alteration of POSTN secretion.…

Figure 2. S68 phosphorylation promotes TW heterodimerization with E12 but is not required for TW homodimerization. (A) Co-immunoprecipitation (left) of endogenous E12 and TW. Antibodies (mouse origin) used for immunoprecipitation are shown on the top. Non-specific IgG is of the same IgG1 isotype. Inputs were 2% of total proteins used for immunoprecipitation. Western blot was probed with rabbit antibody to detect E12 and TW. Co-immunoprecipitation (right) of endogenous pTW S68 and E12. Immunoprecipitates of pTW S68 were detected with mouse E12 antibody. N/S—non-specific signal. (B) Comparable levels of E12, WT and S68A mutant TW expression confirmed by western blot of T98G cells stably transduced with E12 cloned in LXSH or empty LXSH followed by transduction with pLXSN vector, TW, or TW(S68A). (C) Differential detection of E12 after TW immunoprecipitation from nuclear extracts of T98G cells expressing WT or S68A TW. IgG- nonspecific antibody control. (D) Comparable levels of TW and TW(S68A) protein expressed in T98G cells transduced with an empty NTAP-LXSN vector, TW-, or TW(S68A)-NTAP cloned in pLXSN. (E) Enrichment of endogenous E12 in T98G cells expressing WT TW-NTAP versus TW(S68A)-NTAP…

Figure 3. Effect of TW dimer motifs and S68 phosphorylation on POSTN expression. (A) Confirmation of FDC expression in T98G whole cell lysates. Parallel membranes were probed with E12 (top) or TW (middle) antibodies. (B) Marked increase of POSTN mRNA expression in T98G cells expressing TW:E12 heterodimer or TW monomer versus LXSN vector control (* p < 0.0001). (C) Inhibition of POSTN mRNA expression in TW:TW homodimer expressing cells compared to LXSN control (* p < 0.0001). mRNA levels were measured by qRT-PCR. (D) Effect of TW:E12 versus TW (S68A):E12 FDCs on secreted POSTN in T98G cells. FDC expression is confirmed using Tw and E12 antibodies. Secreted POSTN is detected after 24 h in serum free cell culture media and normalized by cell number. Independent experiment was performed to detect TW heterodimers and pTWS68 phosphorylation in WT and S68A mutant FDCs using total TW antibody or pTW S68 antibody, respectively (right panel). (E) Expression of TW homodimers is confirmed using TW antibody. Detection of secreted POSTN and pTW S68 in cells expressing TW:TW, TW(S68S):TW(S68A) or empty vector (LXSN) as described for panel D. (F) Alteration of TW and E12 expression or knockdown results in…

Figure 4. Differential regulation of cellular phenotypes by expression of TW:E12 and TW:TW forced dimer constructs (FDCs). (A) Representative confocal images of FAK immunocytochemistry and phalloidin staining to detect F-actin in T98G cells expressing LXSN control vector, TW:TW and TW:E12 FDCs. FAK localized at membrane (arrows) appears to be increased in TW:E12-expressing cells and co-localize with F-actin filaments. (B) Quantification of FAK at the membrane edge. P values are shown. (C) Cells expressing TW:E12 FDC show increased adhesion to Fibronectin 1 compared to TW:TW FDC or vector control T98G cells. Data shown as fold change over mean adhesion in control accepted as 1. P-values are shown. (D) Dimer dependent activation of pro-invasive proteins. Overexpression of TW:E12 heterodimer results in activation of PDGFRa protein expression and pAKT S473 phosphorylation. Overexpression of TW:TW homodimers results in inhibition of ERK1/2 phosphorylation. Relative expression normalized by actin is shown under the corresponding panel. Expression in cells transduced with empty vector accepted as 1. (E) Matrigel invasion assay showing TW:E12 heterodimer FDC expression markedly increases invasion…

Figure 5. E12 phosphorylation is necessary for regulation of POSTN mRNA expression by TW. (A). Confirmation of specificity of pMAPK/CDK substrate antibody. T98G cells with overexpression WT TW or TW(S68A) mutant were immunoprecipitated with TW antibody or nonspecific IgG. Immunoprecipitated proteins were detected using TW or pMAPK/CDK substrate antibody which recognized only WT TW protein but not the mutant form. (B) The TW:E12 heterodimer is phosphorylated at S139 harboring a PXS*P consensus phosphorylation site. Nuclear extract from T98G cells expressing TW:E12, TW (S68A):E12, and vector were precipitated using TW antibody. Immunoprecipitated proteins were subjected to Western blot analysis and probed with pMAPK/CDK substrate antibody (left) or TW antibody (right). (C) To directly confirm E12 S139 phosphorylation we used cells with TWS68A:E12 or TWS68A:E12S139A dimer overexpression. Proteins immunoprecipitated with TW were probed as in (B). IgG: Nonspecific antibody. CL: Whole cellular lysate used as a 2% input control. β-Actin is not shown. (D). Comparable levels of expression of wild-type and mutant forms of TW:E12 heterodimers in T98G cells after infection with retroviruses harboring…