Tyrosine-phosphorylated galectin-3 protein is resistant to prostate-specific antigen (PSA) cleavage

Abstract

Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer.

FIGURE 1.

Phosphorylation of galectin-3 on Tyr-107 regulates its cleavage by PSA. Panel I, phosphorylation on Tyr-107 (pY107) blocks the cleavage of Gal-3 by PSA in vitro. An equal amount of recombinant galectin-3 wild type was loaded on the gel. Lane 1, galectin-3. Lane 2, PSA-treated galectin-3. Lane 3, galectin-3 was phosphorylated by c-Abl and treated with PSA. The reaction was stopped by adding sample buffer, resolved on a 10% SDS-PAGE gel, and immunoblotted using anti-galectin-3 antibody (A) or anti-Tyr(P) (pTyr) antibody (B). Panel II, tyrosine phosphorylation of galectin-3 in vivo is blocked by c-Abl inhibitor. PC3M cells were treated with 150 ng/ml EGF and 50 ng/ml PDGF for 8 h. One plate was also treated with 1 μm AMN107 (c-Abl inhibitor). Conditioned media were collected, and galectin-3 was immunoprecipitated with TIB166 antibody. Samples were resolved using 10% SDS-PAGE gel and immunoblotted with anti-phosphotyrosine antibody (top panel) anti-galectin-3 (HL31) (middle panel). The bottom panel represents overlapping Tyr(P) and galectin-3 blots loaded and run on an SDS-PAGE gel. Panel III, EGF treatment blocks cleavage of galectin-3 in conditioned medium of co-cultured LNCaP and PC3M cells. LNCaP and PC3M cells were co-cultured for 24 h. One plate was treated with 150 ng/ml EGF for 8 h. Conditioned medium was collected and concentrated using Millipore filters with a 3-kDa cutoff. 50 μg of total protein was loaded and run on a gradient (4–20%) SDS-PAGE gel. After transfer, membrane was blotted with polyclonal HL31 antibody, which can recognize multiple epitopes on galectin-3, for 2 h at room temperature. Panel IV, docking of Gal-3 phosphorylated on Tyr-107 with PTEN. Autodock4 (Scripps Research Institute) was used to dock the phosphorylated version of Gal-3 and PTEN. His-123, Cys-124, Asp-92, and His-93 represent amino acids from the catalytic active site and form the PTEN HCXXGXXR motif, the Cys-124 and Arg-130 residues are essential for catalysis, and the His-123 residue is important for the conformation of the P loop. Panel V, dephosphorylation of galectin-3 Tyr(P)-107 with PTEN. Lane 1, wild type recombinant galectin-3 phosphorylated with c-Abl. Lane 2, phosphorylated galectin-3 treated with PTEN for 2 h at 30 °C. Lane 3, recombinant untreated galectin-3. the top panel represents 15% of the sample mixture run on 10% SDS-PAGE and visualized with Coomassie Blue stain. The rest of the samples were resolved using 10% SDS-PAGE gel and immunoblotted with anti-phosphotyrosine antibody (second panel) and anti-galectin-3 (HL31) antibody (third panel). The bottom panel represents overlapping Tyr(P) and galectin-3 blots. The normalized integrated intensity was calculated as band integrated intensity. AU, arbitrary units.

FIGURE 2.

Functional significance of galectin-3 cleavage by PSA. A, the effect of recombinant full-length galectin-3 and 1–107 and 108–250 fragments on endothelial cell morphogenesis. Three-dimensional heterotypic co-cultures of BAMEC and LNCaP cells on Matrigel were performed in the presence of 10 μg/ml recombinant galectin-3 and its fragments. B, quantitative evaluation of tube formation assay. C, chemotaxis assay in LNCaP cells. Full-length galectin-3 (10 μg/ml) increases chemotaxis in LNCaP cells as compared with fragments of galectin-3 and control cells. Data points show the mean ± S.E. (n = 3) in each condition. D, prostate cancer cells cell migration in the scratch assay. The assay was performed with LNCaP, Du145, and PC3M cell lines. The scratch assay was done to assess in vitro migration of cultured prostate cancer cells stimulated by full-length galectin-3, galectin-3 1–107, and galectin-3 108–250. A uniform wound was made in each plate using a 1-μl pipette tip. The wound area was observed immediately, and at 24 h after creation, cells were counted. Cells were grown under identical conditions. E, a pathway graphic describing the possible mechanisms for the roles of PSA-resistant galectin-3 in the tumorigenesis and progression of prostate cancer.