Vimentin and Non-Muscle Myosin IIA are Members of the Neural Precursor Cell Expressed Developmentally Down-Regulated 9 (NEDD9) Interactome in Head and Neck Squamous Cell Carcinoma Cells

Abstract

Here we demonstrate an interaction between neural precursor cell expressed, developmentally-downregulated 9 (NEDD9) and the cytoskeletal proteins vimentin and non-muscle myosin IIA (NMIIA), based on co-immunoprecipitation and mass spectrometric sequence identification. Vimentin was constitutively phosphorylated at Ser56 but vimentin associated with NEDD9-was not phosphorylated at Ser56. In contrast, NMIIA bound to NEDD9 was phosphorylated on S1943 consistent with its function in invasion and secretion. Treatment of cells with the vimentin-targeting steroidal lactone withaferin A had no effect on vimentin turnover as previously reported, instead causing NEDD9 cleavage and cell death. The NMIIA-selective inhibitor blebbistatin induced cells to form long extensions and attenuated secretion of matrix metalloproteinases (MMPs) 2 and 9. While the site of vimentin interaction on NEDD9 was not defined, NMIIA was found to interact with NEDD9 at its substrate domain. NEDD9 interactions with vimentin and NMIIA are consistent with these proteins having roles in MMP secretion and cell invasion. These findings suggest that a better understanding of NEDD9 signaling is likely to reveal novel therapeutic targets for the prevention of invasion and metastasis.

Figure 1

Identification of vimentin as a NEDD9 interacting protein. A) Analysis of EGFP-NEDD9 immunoprecipitates by mass spectrometry revealed the presence of vimentin. B) Primary sequence of vimentin with Ser56, a common site of phosphorylation in vimentin, shown in red , , . Vimentin present in NEDD9 co-immunoprecipitates was not phosphorylated on Ser56. Based on the peptides identified, 45% of the vimentin sequence was covered in these experiments and is shown in blue.

Figure 2

Validation of the interaction between NEDD9 and vimentin. A) (Left), UMSCC22A and UMSCC22B were immunoblotted for NEDD9, vimentin, MICAL1 and GAPDH. UMSCC22A cells, isolated from a primary tumor of the tongue, lack vimentin expression whereas UMSCC22B cells, derived from a metastasis in the same patient, exhibit vimentin expression. NEDD9 and the NEDD9 and vimentin interacting protein MICAL1 were expressed in both cell lines. (Right), Vimentin co-immunoprecipitates with NEDD9 in UMSCC22B and SCC25 cells. B) Vimentin co-immunoprecipitates with NEDD9 in SCC9 cells, but not in UMSCC22A cells which lack vimentin expression. Vimentin co-immunoprecipitates with the NEDD9-interacting protein MICAL1 in SCC9 cells . C) Fractionation of SCC9 cells demonstrates that vimentin preferentially associates with the nuclear/cytoskeletal fraction where it associates with NEDD9. D) Confocal microscopy confirms the perinuclear and cytoplasmic distribution of vimentin.

Figure 3

Withaferin-A (WFA) treatment results in NEDD9 cleavage and cell death. A). Immunoblot of control, DMSO and WFA (5 μM) treated SCC9 cells. B) NEDD9 structure showing location of its caspase cleavage sites DLVD and DDYD. C) Dose-dependent change in SCC9 cell morphology (upper) in response to WFA treatment and cleavage of NEDD9 (lower). The WFA-induced loss of NEDD9 via caspase cleavage is consistent with apoptotic cell death. Also shown is loss of MMP9/2 secretion in response to WFA treatment, reflecting cell death.

Figure 4

Vimentin is a phosphoprotein. A) Schematic of vimentin structure delineating its head, α-helical and tail domains , . The head domain is the principal site of of phosphorylation by a number of kinases, which in turn, regulates vimentin filament assembly/dis-assembly. Ser56 phosphorylation causes disassociatiuon of the NEDD9 paralog, p130Cas from vimentin , . B) Ser56 phosphorylation of vimentin in control and growth factor treated SCC9 cells. C-E) Ser38 phsophorylation of vimentin in response to growth factor treatment with quantification of the response to growth factors.

Figure 5

Identification of NMIIA as a NEDD9 interacting protein. A) Analysis of EGFP-NEDD9 immunoprecipitates by mass spectrometry revealed NMIIA as a NEDD9 binding partner. Ser1943 of NMIIA was found to be phosphorylated consistent with its role in regulating NMIIA localization and filament assembly , .

Figure 6

Inability of substrate domain to bec phosphorylated eliminates NMIIA-NEDD9 binding interaction. NMIIA was immunoprecipitated from a panel of cell lines stably expressing the EGFP-tagged NEDD9 mutants indicated. The presence of NEDD9 in the immunoprecipitates was evaluated by immunoblotting with antii-GFP antibodies. Shown in the upper image is the structure of NEDD9 with each of its domains indicated; SH3 domain (SH3), substrate domain (SD), serine-rich region (SRR) and C-terminal focal adhesion targeting domain (FAT). The lower image is a representative gel demonstrating the results of NMIIA immunoprecipitation followed by GFP immunoblot. The red dots depict the migration position of the NEDD9 mutant being blotted. Also evident are two non-specific bands also detected in the blank and empy vector control lanes.

Figure 7

NMIIA expression in HNSCC cell lines and presence in the cytoskeletal fraction. A) NMIIA expressed in HNSCC cell lines. B) NMIIA is present in nuclear and cytoplasmic fractions. C) Treatment of cells with blebbistatin decreases the association of NMIIA with the nuclear/cytoplasmic fraction. D) Confocal microscopy of control and blebbistatin-treated SCC9 cells.

Figure 8

Evaluation of NMIIA's role in MMP9/2 secretion. A) SCC9 cells grown in SFM were treated with the inhibitor or growth factor indicated for 18 h. Aliquots of the conditioned medium were examined by in-gel zymography. B) Analysis of MMP2 and MMP9 secretion shown in A. C) Light microscopic examination of the cells treated as indicated in A. These data are representative of three independent experiments.