Phosphatase of regenerating liver 3 (PRL3) provokes a tyrosine phosphoproteome to drive prometastatic signal transduction

Abstract

Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the "PRL3-mediated signaling network." Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for "hijacking" this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation.

Figures

Fig. 1.

Ectopic PRL3 expression induces aberrant regulation of tyrosine phosphorylation.A, PRL3 transcript is significantly enhanced in the PRL3-WT and -C104S (phosphatase dead) expressing HEK293 clones, relative to endogenous levels of PRL3 transcript in vector counterparts as observed through RT-PCR using PRL3-specific oligonucleotides. 18S-rRNA was used as control. RFP (red fluorescent protein)-tagged PRL3-WT protein is localized on endomembranes following stable ectopic expression in HEK293 cells as assessed by confocal microscopy. B, PRL3-WT cells have an unmistakable “spindle-like” fibroblast/mesenchymal morphology as compared with their “squamous” epithelial PRL3-C104S “phosphatase-dead” and vector counterparts. C, PRL3-WT clones have enhanced “global” tyrosine phosphorylation (“pan” pTyr-α), a markedly less latent pTyr527-Src population (pTyr527-α), and constitutive phosphorylated/activated ERK1/2 (pThr202/pTyr204-α) and STAT3 (pTyr705-α), relative to vector counterparts as measured by phospho-specific immunoblotting.

Fig. 2.

Phosphoproteomic methodology. Data acquisition and analysis flow-chart describing the sample preparation and analysis methodology for both qualitative and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative assessment of tyrosine phosphorylation.

Fig. 3.

Quality of mass spectra used for SILAC-based quantitative assessment of tyrosine phosphorylation. Representative fragmentation spectra for phosphopeptides representing pTyr187 of ERK2 (173VADPDHDHTGFLTEY[PO32-]VATR), pTyr705 of STAT3 (K.686YCRPESQEHPEADPGSAAPY[PO32-]LK), and pTyr417 of PAG1 (414ENDY[PO32-]ESISDLQQGR) showing the quality of mass spectra used for SILAC-based quantitative assessment of tyrosine phosphorylation. Raw fragmentation “sequencing” spectra including parent ion abundances (SILAC-based quantitative spectra: SILAC-“Light” (vector; blue points); SILAC-“Heavy” (PRL-3; red points)). b-ion series is colored in “red” and y-ion series in “blue.”

Fig. 4.

Ectopic PRL3 expression induces aberrant activation of mitogenic and chemotactic signal transduction.A, Phosphoproteomic study summary in pie-chart format (sub-categories comprising each bio-functional bin can be seen in the entire curated dataset represented in supplemental Table S1). 75% of phosphoproteins are unique to the PRL3 expressing cells and concentrate within “Cellular Communication and Signal Transduction.” B, Ingenuity Pathway Analysis (IPA) results showing the Top 10 biofunctions and canonical pathways predicted from the phosphoproteomic data set. Data significance is represented using a B-H (Benjamini-Hochberg) p value adjustment to the false-discovery-rate (FDR; q-value). Phosphoproteomic data provide evidence in support of mitogenic and chemotactic signal transduction being prominently affected following ectopic PRL3 expression including significant representation of networks associated with PDGF, Neuregulin, p21/Cdc42/Rac1-activated kinase (Pak), Ephrin (Eph) receptor, Interleukin, and Integrin signaling.

Fig. 5.

The PRL3-mediated signaling network. Graphical model depicting phosphoproteomic data present within the PRL3 dataset that supports a mechanism by which PRL3 potentiates pro-metastatic signal transduction by exploiting “oncogenic” Src kinase activity. Src activates a signal transduction network coordinated by a mitogenic and chemotactic PDGF (α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array in the PRL3 cells. Proteins highlighted within green ovals represent phosphoproteins shown by both qualitative and SILAC-based quantitative mass analysis to be either significantly up-regulated (pTyr-residue; blue circles) or exclusively present (pTyr-residue; red circles) within the PRL3 data set. Nonhighlighted proteins represent members of canonical signal transduction pathways assumed to be activated based on data present within the PRL3 phosphoproteomic dataset. pTyr-residue number designation is based on documented (NP and GI numbers) in supplemental Table S1.

Fig. 6.

Ectopic PRL3 expression induces selective expression and/or stabilization of the PDGFβ-receptor and Src-dependent constitutive tyrosine phosphorylation of the PDGFβ-receptor and PLCγ1.A, The PDGFβ-receptor is selectively expressed and/or stabilized and is constitutive phosphorylated in the PRL3 cells as assessed by PDGFRβ antibody-specific immuoprecipitation (IP) followed by “pan”-pTyr immunoblotting. PDGFRβ phosphorylation is dependent on the activity of the Src kinase as assessed by the use of the Src kinase chemical inhibitor (SU6656) at 2.5 μm for 2h. B, Phospholipase-C gamma 1 (PLCγ1) is constitutive phosphorylated in the PRL3 cells as assessed by phosphotyrosine-specific immunoblotting against pTyr-783. Phosphorylation of PLCγ1 on Tyr-783 is dependent on the activity of the Src kinase. The attenuation of ERK1/2 phosphorylation using SU6656 is used as a positive control for SU6656-mediated Src kinase inhibition based on our previous published results (14). C, Data validation and spectral quality for pTyr783-PLCγ1 (779NPGFY[PO32-]VEANPMPTFK): Raw fragmentation sequencing spectra including parent ion abundances (SILAC-based quantitative spectra: SILAC-Light (vector; blue points); SILAC-Heavy (PRL-3; red points)). b-ion series is colored in red and y-ion series in blue.