Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition

Abstract

Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin-dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632-638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin-mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.

Figure 1.

ERK/MLCK activities are required for Src-induced integrin adhesion assembly in KM12C colon cancer cells. (A) KM12C cells transfected with Src527F (KM12C/Src527F) were cultured in uncoated plastic dishes (adherent) or in poly-HEMA–coated dishes (suspension). Phospho-ERK or phospho-MLC levels were detected by probing total lysates with the anti-phospho-ERK (Thr202/Tyr204) or anti-phospho-MLC (Ser19) antibodies (top panels). The filters were reprobed with anti-ERK or anti-MLC antibodies (lower panels). (B) Increased level of total cellular phospho-MLC in KM12C/Src527F cells was blocked by MLCK inhibitor ML9 (7.6 μM). (C) (a–f) KM12C/Src527F (a–c) or KM12C/vector (d–f) cells were plated on fibronectin-coated substratum for 6 h, fixed, and stained with anti-paxillin (a and d), anti-phospho-ERK (Thr202/Tyr204; b and e) or anti-phospho-MLC (Ser19; c and f) antibodies. Arrows show paxillin, phospho-ERK and phospho-MLC localized at cell-matrix adhesion complexes at the ends of protrusive structures in KM12C/Src527F cells. (g–m) MEK inhibitor UO126 (25 μM; g–i) or MLCK inhibitor ML7 (5 μM; j–m) blocked the formation of protrusive integrin-mediated adhesions in KM12C/Src527F cells. Arrows show localization of paxillin or phospho-ERK at nonprotrusive cell-matrix adhesion structures at the cell periphery. Scale bars, 10 μm. (D) KM12C/Src527F cells were plated on poly-l-lysine–coated substratum, fixed, and stained with anti-paxillin (a), anti-phospho-ERK (Thr202/Tyr204; b) or anti-phospho-MLC (Ser19; c) antibodies. Scale bars, 10 μm.

Figure 2.

Serum induces peripheral integrin adhesion structures in KM12C cells via MEK/ERK and MLCK activities. (a--d) KM12C/vector cells were switched to serum-free low-calcium medium (KGM) for 24 h, fixed, and costained with anti-FAK and anti-phospho-MLC (Ser19; a and b) or anti-FAK and anti-phospho-ERK (Thr202/Tyr204) (c and d) antibodies. Scale bars, 10 μm. (e–h) Serum-starved KM12C/vector cells were stimulated with MEM containing 10% FBS for 10 min, fixed, and costained with anti-FAK and anti-phospho-MLC (Ser19) (e and f) or anti-FAK and anti-phospho-ERK (Thr202/Tyr204) antibodies (g and h). Arrows in e indicate the formation of peripheral focal adhesion complexes after stimulation with serum. Arrows in f show serum-induced accumulation of phospho-MLC at the cell periphery. Scale bars, 10 μm. (i–m) KM12C/vector cells were treated as described above except that stimulation with serum was carried out in the presence of ML9 (7.6 μM; i and j) or UO126 (25 μM; l and m). Cells were fixed and costained with anti-FAK and anti-phospho-MLC (Ser19) (i and j) or anti-FAK and anti-phospho-ERK (Thr202/Tyr204) antibodies (l and m). Scale bars, 10 μm.

Figure 3.

SH2 and SH3 domains of Src are required for accumulation of phospho-MLC at the cell periphery. (A) Expression levels of mutant Src kinase proteins in KM12C cells. (B) KM12C/Src527F, KM12C/Src527F/R175L, or KM12C/Src527F/W118A cells were plated on fibronectin for 6 h (a–c) or cultured in MEM and switched to low-calcium medium (d–f). Cells were fixed and stained with anti-phospho-Src (Tyr416) antibody. Scale bars, 20 μm. (C) Phospho-MLC levels in KM12C/vector, KM12C/Src527F, KM12C/Src527F/R175L, or KM12C/Src527F/W118A cells were detected by probing total lysates with the anti-phospho-MLC (Ser19) antibody (top panel). The filters were reprobed with anti-MLC antibody (bottom panel). (D) KM12C/Src527F, KM12C/Src527F/R175L, or KM12C/Src527F/W118A cells were switched to low-calcium medium, fixed, and stained with anti-paxillin (a–c) or anti-phospho-MLC (Ser19) (d–f) antibody. Arrows in d show accumulation of phospho-MLC at the cell periphery. Scale bars, 20 μm. (E) Vimentin expression levels in KM12C/vector, KM12C/Src527F, KM12C/Src527F/K295M, KM12C/Src527F/R175L, or KM12C/Src527F/W118A cells.

Figure 4.

Src kinase activity at peripheral integrin-mediated adhesion complexes is required to impair E-cadherin localization. (A) KM12C cells expressing Src527F (a), Src527F/R175L (b), Src527F/W118A (c) or kinase-defective Src527F/K295M) (d) were switched from low- to high-calcium medium for 4 h. Cells were fixed and stained with anti-E-cadherin. Scale bars, 20 μm. (B) Quantitation of percentage of cells which localize E-cadherin to cell-cell contacts after switch to high-calcium medium. The mean and range of two independent experiments is shown.

Figure 6.

Inhibition of MEK/ERK, ROCK, and MLCK activities reverses Src-induced deregulation of E-cadherin–associated cell-cell contacts. (A) KM12C/vector or KM12C/Src527F cells were switched from low- to high-calcium medium for 4 h (a and b). MEK inhibitor UO126 (25 μM; c), ERK activation inhibitor peptide II (100 μM; d), ROCK inhibitor Y27632 (10 μm; e), or MLCK inhibitor ML7 (5 μM; f) was added to high-calcium medium and KM12C/Src527F cells were maintained for 4 h in such medium. Cells were fixed and stained with anti–E-cadherin antibody. Solid arrows show accumulation of E-cadherin at cell-cell contacts in KM12C/vector cells and KM12C/Src527F cells treated with either UO126, ERK activation inhibitor peptide II, Y27632, or ML7 (a, c, and d–f). Broken arrow in b points to disrupted E-cadherin staining in KM12C/Src527F cells after the switch to high-calcium medium. Scale bars, 10 μm. (B) Quantitation of percentage of KM12C/Src527F cells that are forming cadherin-mediated cell-cell contacts when cells are switched to high-calcium medium containing ML7, UO126, or Y27632. (C) KM12C/Src527F were plated on fibronectin-coated substratum for 2 or6h(a–f) without inhibitors (a and d) or with the MEK inhibitor UO126 (25 μM; b and e) or MLCK inhibitor ML7 (5 μM; c and f). Cells were fixed and stained with anti–E-cadherin antibody. Arrows show accumulation of E-cadherin at cell-cell contacts in KM12C/Src527F cells plated on fibronectin when MEK/ERK or MLCK activity is inhibited. Scale bars, 10 μm. (D) (a) KM12C/Src527F were plated on poly-l-lysine–coated substratum for 6 h and E-cadherin was visualized by staining with anti–E-cadherin antibody. Arrows indicate accumulation of E-cadherin between active Src527F expressing cells when integrin signaling is suppressed. (b) KM12C/vector cells were plated on fibronectin for 6 h, fixed, and stained with anti–E-cadherin antibody. Arrows show accumulation of E-cadherin at cell-cell contacts. Scale bars, 10 μm.

Figure 5.

Inhibition of Src kinase, MEK/ERK, ROCK, or MLCK activity impairs localization of phospho-MLC at the cell periphery. KM12C/Src527F cells were cultured in high-calcium medium (MEM), further maintained in low-calcium medium and treated with either PP2 (20 μM; b), UO126 (25 μM; c), Y27632 (10 μM; d), or ML7 (5 μM; e) for 4 h. Cells were fixed and stained with anti-phospho-MLC (Ser19) antibody. Arrows show phospho-MLC localization at the leading edge of protrusive adhesion structures. Scale bars, 10 μm.

Figure 7.

Inhibition of PI 3-kinase or STAT3 activity does not induce cell-cell contact formation in active Src-expressing cells. (A) KM12C cells expressing active Src527F were treated with the PI 3-kinase inhibitor LY294002 (50 μM). Cell lysates were probed with anti-phospho-Akt (Ser473) or anti-Akt antibody (left panels). Levels of active STAT3 (phospho-Tyr705) and total STAT3 in KM12C/Src527F cells or A431 cells stimulated with EGF are shown in the right panels. (B) Percentage of KM12C/Src527F cells that are forming cadherin-mediated cell-cell contacts after the cells are switched to high-calcium medium without inhibitors or with ERK activation inhibitory peptide II (100 μM), STAT3 inhibitory peptide (100 μM), or PI 3-kinase inhibitor LY294002 (50 μM).