Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

Abstract

Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1-clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.

Fig 1. Expression of profilin 1 mutants in PC-3 cells.

(A) Scheme of the DNA construct of profilin 1 mutants. (B) Restriction analysis of the DNA construct ligated into the pcDNA3 cloning vector using EcoRI and BamHI. Line 1: GeneRuler 1 kb DNA Ladder (Promega), line 2: restriction of Pfn1-Tyr139 mutant, line 3: restriction of Pfn1-Q138P mutant. (C) Western blot analysis of transfected PC-3 cell lysates expressing different profilin 1 mutants. Cell lysates were prepared 48 h post transfection. Line 1: size marker (SeeBlue® Pre-stained Protein Standard, Life Technologies), line 2: transfected empty pcDNA3 vector, line 3: transfected Pfn1-Tyr139/pcDNA3, line 4: transfected Pfn1-Q138P/pcDNA3. Mutants were detected with anti-FLAG antibodies. Detection of profilin 1 (native and mutated) and β-actin on the same membrane is also shown. Due to the chemiluminescent detection, size marker is added as a separate strip.

Fig 2. Migration and invasion of PC-3 cells transfected with profilin1 mutants.

The level of cell migration/invasion, assessed by increases in the curve slopes (1/h), is shown. All results were normalized to cells transfected with empty pcDNA3 vector (the control). Assays were carried out in triplicate. (A) Results from migration were calculated for the interval between the 45 hour and 98 hour time points, during which the slopes of the curves were linear. (B) Results from invasion were calculated for the interval between the 49 hour and 107 hour time points. (C) The influence of AMS36 inhibitor (10 μM) and recombinant cathepsin X (2 μM) was tested on the cells transfected with Pfn1-Tyr139 mutant. Results of migration were calculated for the interval between the 29 hour and 38 hour time points. *P≤0.05 **P<0.01; ***P<0.001.

Fig 3. Presence of the profilin 1—clathrin complex is cathepsin X dependent.

Cells were treated with cathepsin X inhibitor AMS36 (10 μM) or DMSO. Profilin 1—clathrin complexes were detected with a proximity ligation assay and analyzed with confocal microscopy. Each red dot represents a Texas red signal present on the spot with the complex. Cell nuclei were stained with DAPI. Bar, 20 μm.

Fig 4. Endocytosis of fluorescein-labeled dextran is abrogated by cathepsin X.

Clathrin-mediated endocytosis of fluorescein-labeled dextran (10 kDa) was followed by flow cytometry. Cathepsin X in cells was (A) treated with cathepsin X inhibitor AMS36 (10 μM) or (B) silenced with its specific siRNA. Control cells were treated with DMSO or transfected with control siRNA. (C) Prior to the inhibitor treatment, cells were treated with 10 μM chlorpromazine (CHL). Median values of two or three independent experiments (each in triplicate) are shown. *P<0.05; **P<0.01 (D) Endocytosis of fluorescein-labeled dextran was followed under the microscope. Representative examples of DMSO and AMS36 treated cells are shown. (E) Areas and mean intensity values of the cells were measured using ZEN 2012 software and the mean intensity/1000 μm² of the cell area calculated. 28 (DMSO) and 18 (AMS36) cells were measured. Bars, 5 μm.

Fig 5. Endocytosis of transferrin Alexa Fluor 555 conjugate is abrogated by cathepsin X.

Clathrin-mediated endocytosis of transferrin Alexa Fluor 555 conjugate was followed by flow cytometry. Cathepsin X in cells was silenced with its specific siRNA, control cells were transfected with control siRNA. Mean values of three independent experiments (each in duplicate) are shown. In a control experiment transferrin receptor was saturated with holo-transferrin. *P<0.05.

Fig 6. Profilin 1 mutant Pfn1-Tyr139 decreases endocytosis and actin polymerization.

(A) Cells were transfected with two plasmids carrying different profilin 1 mutants and empty plasmid (control). Clathrin-mediated endocytosis of fluorescein-labeled dextran (10 kDa) was followed with flow cytometry. Median values are representative of two independent experiments (one in triplicate and one in duplicate). *P<0.05 (B) Cells were transfected with different profilin 1 mutant constructs. After 48 hours filamentous actin was stained with phalloidin conjugate and analyzed with flow cytometry. Mean values are representative of two independent experiments (one in triplicate and one in duplicate). *P<0.05.