Figure 1.
Identification of CIN85 and CD2AP as LOX-PP interacting proteins in breast cancer cells.
(A) Extracts from ZR-75 cells transfected with vectors expressing GST or LOX-PP-GST were precipitated with Glutathione-Sepharose 4B beads, resolved by SDS-PAGE and silver stained. The band(s) at ∼85 kDa was analyzed by LC-MS/MS mass spectrometry and identified as CIN85 and CD2AP. *, non-specific proteins. The positions of the co-precipitated CD2AP/CIN85 and LOX-PP proteins are indicated by the solid and large hatched arrows, respectively, and of GST by the dashed line. (B–C) GST or LOX-PP-GST (PP-GST) was co-expressed with GFP-CIN85 WT (B) or FLAG-CD2AP (C) in HEK293T cells, and LOX-PP associated proteins isolated by GST-pull down assays and subjected to WB for GFP (B) or FLAG (C) and GST. Input, 4% of lysates (4%). (D) Recombinant LOX-PP-myc-His (0.5 µM) was subjected to a GST-pull down assay using 0.5 µM of either GST or GST (G)-CIN85, and WB for the His or CIN85 (Calbiochem) antibody. Input, 5%. (E) Samples of whole cell extracts (10 µg) of the indicated human and mouse cells were subjected to WB for CIN85 (Upstate). (F) (Left) TX-100 extracts of Hs578T (Upper) or ZR-75 (Lower) cells were immunoprecipitated with mouse IgG or CIN85 (Upstate) antibody, and analyzed for CIN85 (Upstate) and LOX-PP. (Right) TX-100 extracts of Hs578T (upper) or ZR-75 (lower) cells were immunoprecipitated with rabbit IgG or LOX-PP antibodies, and subjected to WB.
Figure 2.
LOX-PP reduces CIN85 mono-ubiquitination and ability to interact with c-Cbl.
(A) GST or LOX-PP-GST (PP-GST) was expressed in ZR-75 (left panel) or Hs578T (right panel) cells. GST and associated proteins were precipitated as described in Fig. 1B and subjected to WB for CIN85 (Upstate Biotechnology and Calbiochem antibodies for ZR-75 and Hs578T cells, respectively), CD2AP (H-290), c-Cbl, AMAP1, EGFR, p130Cas and GST. Input, 4%. (B–C) HEK293T cells were transfected with AMAP1-FLAG, CIN85, HA-c-Cbl, HA-ubiquitin and LOX-PP-GST (PP-GST) as indicated and subjected to a ubiquitination assay. FLAG-tagged AMAP1 was immunoprecipitated and total whole cell extracts were subjected to WB with an HA antibody (upper panel), or the indicated antibodies (lower panel). (B). Data were quantified and relative mono-ubiquitination of AMAP1 with and without LOX-PP was determined by averaging the results of three independent experiments (C). P value was calculated using Student's t-test. *, P<0.03. (D) FLAG-CIN85 was expressed in Hs578T (left panel) or MCF-7 (right panel) cells. After lysis, the indicated amount of recombinant LOX-PP-myc-His was added and the mixture incubated at 4°C for 2 h. Proteins were then immunoprecipitated with a FLAG antibody and subjected to WB with FLAG and c-Cbl antibodies. (L Exp, longer exposure).
Figure 3.
The SH3-B domain of CIN85 interacts with the LOX-PP region preceding aa 120.
(A) Schematic representation of the full length CIN85 (WT) and the amino terminal-containing deletion mutant (NT). The SH3, Src homology 3 domains A, B, and C, PR, proline-rich region, CC, coiled-coil domain are indicated. (B) GST or LOX-PP-GST (PP-GST) protein was co-expressed with FLAG-tagged CIN85 WT or CIN85 NT in HEK293T cells. GST-pull down assays were performed and bound or whole cell extracts subjected to WB with FLAG and GST antibodies. (C) Recombinant LOX-PP-myc-His (0.5 µM) was incubated with GST, GST (G-)-tagged SH3-A, SH3-B or SH3-C peptides (0.5 µM each) and subjected to a GST-pull down assay. The precipitated proteins were analyzed by Coomassie staining (lower panel) and with antibodies against LOX-PP (upper panel). (D) Schematic representation of LOX-PP deletion mutants prepared in the pcDNA3-GST vector. SP, Signal peptide. The lengths of the constructs are indicated on the left. (E–F) HA-CIN85 was co-transfected with either LOX-PP-WT (1-162) or the indicated deletion mutants (from part D) or empty vector (EV) DNA into HEK293T cells. Extracts were prepared and samples (4%) were analyzed directly as a measure of input (E) or subjected to GST-pull down assays and WB for HA and GST (F). (G) Amino acid sequences of LOX-PP from various species are shown. The positions of aa 111 and aa 116 and of aa 113 and aa 117 are indicated by black and grey boxes, respectively.
Figure 4.
Alanine scanning mutagenesis reveals amino acids of LOX-PP required for CIN85 binding and c-Cbl competition.
(A) To begin to identify the critical amino acids in LOX-PP, vectors expressing individual point mutants in which residues aa 111 to aa 120 were replaced with alanine, or WT LOX-PP protein were co-transfected with FLAG-CIN85 WT in HEK293T cells. (right panel) The mutated proteins compared with the WT LOX-PP for their ability to interact with CIN85 using GST-pull down assays and WB with FLAG and GST antibodies. (left panel) Input, 4%. EV, empty vector DNA. (B) To confirm the interaction of CIN85 and endogenous c-Cbl, GFP or GFP-CIN85 proteins were expressed in HEK293T cells and TX-100 extracts prepared. Following immunoprecipitation with a GFP antibody, the precipitated proteins subjected to WB with antibodies against c-Cbl (upper panel) and GFP (lower panel). (C) To test whether LOX-PP mutants unable to interact with CIN85 compete for its binding with c-Cbl, GFP-tagged-full-length CIN85 was co-expressed with GST, or GST-tagged LOX-PP-WT (PP-WT-G), LOX-PP-P111A (PP-P111A-G), or LOX-PP-R116A (PP-R116A-G) in HEK293T cells. TX-100 extracts were immunoprecipitated with GFP antibody and the precipitated proteins detected with antibodies against c-Cbl, GFP and GST. Input, 4%. (D) To test the specificity of the changes in binding of the mutant LOX-PP proteins, vectors expressing GST tagged LOX-PP-WT (WT), or mutants LOX-PP-P111A (P111A) or LOX-PP-R116A (R116A) or GST (EV) were transfected into ZR-75 cells and their ability to interact with c-Raf, which maps to aa 26-100, monitored by GST-pull down assays. WB for CIN85 (Upstate: clone 84), c-Raf and GST was performed.
Figure 5.
Structural model of CIN85 SH3-B domain in complex with the LOX-PP peptide. Upper part:
Front and rear views of the modeled SH3-B (pale green) and LOX-PP (yellow sticks) complex are depicted; selected, key electrostatic interactions are represented by blue dashed lines. The open arrow (upper right) indicates the approximate position of Arg(-2) in structures that can assemble the ternary complex: SH3–[RxP1xxxxR/K peptide]–SH3. Inset box: Detail showing the structural dissimilarity in the n-Src loop between CIN85 SH3-B (our model) and SH3-A, superimposed (PDB code: 2BZ8; brown cartoon). In the SH3-A structure, the bound Cbl-b peptide backbone (light brown) is also seen to diverge more towards its C-terminus from the LOX-PP peptide position. Lower part: Comparison of the electrostatic potential surfaces of SH3 domains A and B in equivalent orientations, illustrating their differential charge distributions. The Adaptive Poisson-Boltzmann Solver (APBS) software [55] was used to generate the electrostatic potential map, contoured in varying colour intensity from -15 (red) through 0 (white) to +15 (blue) kT/e, and rendered within PyMOL (www.pymol.org).
Figure 6.
CIN85 promotes degradation of the extracellular matrix.
NF639 and MDA-MB-231 breast cancer cells were transfected with 20 nM of either siCIN85 or scrambled negative control siRNA (siControl). (A) After 48 h, samples of whole cell extracts (10 µg) were subjected to WB for CIN85 (Upstate: clone84) and β-actin. (B) Alternatively, cells were plated on coverslips coated with FITC-conjugated gelatin and incubated for 4 h. Cells were fixed 3.7% paraformaldehyde. F-actin and nuclei were labeled with TRITC-phalloidin (red) and Hoechst 33342 (blue), respectively. Lines indicate region of XY image projected to generate orthogonal planes XZ and YZ. Bars: 10 µm.
Figure 7.
LOX-PP mutants unable to interact with CIN85 are unable to inhibit breast cancer cell invasion. Stable transductants of NF639-EV, and NF639-LOX-PP (PP) -WT, -P111A and -R116A cells were prepared.
(A) These were analyzed by WB for LOX-PP expression using the V5 tag. (B,C) Cells were subjected to a collagen degradation assay using coverslips coated with FITC-conjugated gelatin (green). F-actin and nuclei were labeled with TRITC-phalloidin (red) and Hoechst 33342 (blue), respectively and cells photographed at 50x magnification. Bars: 20 µm (B). Alternatively, confocal images of the slides are shown. Lines indicate region of XY image projected to generate orthogonal planes XZ and YZ. Bars: 10 µm (C). (D,E) The indicated NF639 cells were subjected to invasion assays, in triplicate, and photographed (Bar: 0.25 mm) (D). The numbers of cells invaded per field were determined (E).The average data from three independent experiments ± SD is presented relative to the EV control (set at 100%). P values were calculated using Student's t-test. *, P < 0.01 (E). (F) Stable transductants of NF639 cells were subjected to a Matrigel outgrowth assay. After 5 days, slides were photographed at 40x magnification. Bars: 0.25 mm.