Trask phosphorylation defines the reverse mode of a phosphotyrosine signaling switch that underlies cell anchorage state

Abstract

Phosphotyrosine signaling in anchored epithelial cells constitutes a spacially ordained signaling program that largely functions to promote integrin-linked focal adhesion complexes, serving to secure cell anchorage to matrix and as a bidirectional signaling hub that coordinates the physical state of the cell and its environment with cellular functions including proliferation and survival. Cells release their adhesions during processes such as mitosis, migration, or tumorigenesis, but the fate of signaling through tyrosine phosphorylation in unanchored cells remains poorly understood. In an examination of epithelial cells in the unanchored state, we find abundant phosphotyrosine signaling, largely recommitted to an anti-adhesive function mediated through the Src family phosphorylation of their transmembrane substrate Trask/CDCP1/gp140. Src-Trask phosphorylation inhibits integrin clustering and focal adhesion assembly and signaling, defining an active phosphotyrosine signaling program underlying the unanchored state. Src-Trask signaling and Src-focal adhesion signaling inactivate each other, constituting two opposing modes of phosphotyrosine signaling that define a switch underline cell anchorage state. Src kinases are prominent drivers of both signaling modes, identifying their position at the helm of adhesion signaling capable of specifying anchorage state through substrate selection. These experimental studies along with concurring phylogenetic evidence suggest that phosphorylation on tyrosine is a signaling function fundamentally linked with the regulation of integrins.

Figure 1

(A) MCF10A cells were assayed by anti-phosphotyrosine immunoblotting while adherent (A) or after 2 hours culture in suspension in non-adherent ULC plates (S). In lanes 3 and 4 suspended cells were treated with the src-selective inhibitors PP1 and PP2 at 10 uM for 1 hour. (B and C) MCF10A cells were engineered to stably express Trask shRNA or a control non-silencing shRNA and the absence of Trask and pY-Trask was confirmed as shown. (D) Anti-phosphotyrosine immunoblotting shows that shRNA knockdown of Trask removes the predominant tyrosine phosphoprotein of the suspended state. (E) Phosphotyrosine immunoblot analysis of MCF10A cells in the adherent (A) or suspended (S) states (lanes 1 and 2). Lysates from suspended cells were also run after immunodepletion with rabbit anti-Trask antibodies (lane 3) or control IgG (lane 4) to remove Trask protein from the lysate. Arrows indicate the 140 and 80 kD Trask proteins.

Figure 2

Phosphotyrosine immunoblot analysis of a variety of epithelial cell lines in the adherent (A) or suspended (S) states. Arrows indicate the 80 and 140 kd Trask proteins. Lanes correspond to (1) MDA-468 and (2) MDA-231 breast cancer cells, (3) Du145 and (4) PC3 prostate cancer cells and (5) DLD1 and (6) HCT116 colon cancer cells.

Figure 3

MCF10A cells were fixed either while in the adherent state (left) or after being cultured for 2 hours in suspension (right). Cells were fixed in paraformaldehyde, permeabilized in methanol and immunostained with the indicated primary antibodies and Alexa Fluor 546 conjugated secondary antibodies. Cells were visualized under an LSM510 Carl Zeiss confocal microscope, at 630× magnification and images captured using the LSM Image browser.

Figure 4

Cell lysates from the indicated cell lines were immunoprecipitated with rabbit polyclonal anti-Trask antibodies and immunoblotted with mouse anti-Trask or antiphosphotyrosine monoclonal antibodies.

Figure 5

MCF10A cells were grown in matrigel in 3D, fixed and immunostained as indicated at days 3, 7 and 12 after seeding and imaged under confocal fluorescence microscopy using the indicated primary antibodies and Alexa Fluor 546 (red) or Alexa Fluor 488 (green) conjugated secondary antibodies. The pY-Trask (red) and laminin (green) immunostains are shown on the left and the Trask immunostains (green) are shown on the right.

Figure 6

Anti-phospho-Trask immunohistochemical staining of a human colon cancer specimen showing phosphorylation of Trask in regions of the tumor.