PMC

Phospho-p130CAS, vinculin, paxillin, and FAK are enriched in peripheral adhesion complexes. A–I, indirect immunofluorescence of PC3 cells stably expressing Palm-PTK6-YF was performed using anti-p130CAS (A), phospho-p130CAS (PY-664) (B), phospho-p130CAS (PY-165) (C), phosphotyrosine (PY) (D), FAK (E), phospho-PTK6 (PY-342) (G), paxillin (H), and vinculin (I) antibodies. F is a merge of D and E. J–L, PC3 cells expressing vector controls were grown at collagen I-coated chamber slides, and indirect immunofluorescence was performed using anti-phospho-PTK6 (PY-342) (J), phospho-p130CAS (PY-165) (K), phosphotyrosine (PY) (L) antibodies. Cells were counterstained with DAPI (blue). The size bar denotes 20 μm.

Integrin and growth factor signaling promote formation of peripheral adhesion complexes. A, Palm-PTK6-YF-expressing PC3 cells form more peripheral adhesions on collagen I-coated plates than on non-coated plates. PC3 cells stably expressing Palm-PTK6-YF or vector (Vec) were seeded in collagen I-coated or non-coated chamber slides for 24 h. Cells were stained with anti-phosphotyrosine antibodies (green) and counterstained with DAPI (blue). The size bar denotes 50 μm. B, 1-h 20% FBS stimulation after 24-h serum starvation promotes the formation of peripheral adhesion complexes in Palm-PTK6-YF-expressing PC3 cells. Peripheral adhesion complexes were visualized by anti-phosphotyrosine immunostaining (green). Cells were counterstained with DAPI (blue). The size bar denotes 50 μm.

Formation of peripheral adhesion complexes depends on PTK6 kinase activity and membrane localization. A, immunoblot analysis of total cell lysates of PC3 cells stably expressing vector (Vec), Myc-tagged PTK6-KM, PTK6-WT, PTK6-YF, Palm-PTK6-KM, Palm-PTK6-WT, Palm-PTK6-YF, or NLS-PTK-YF was performed with anti-Myc tag and α-tubulin antibodies. B, peripheral adhesion complexes only form in Palm-PTK6-YF- or Palm-PTK6-WT-expressing cells. Phase-contrast images of PC3 cells described in A are shown. The size bar denotes 50 μm. C, induced phosphotyrosine signaling in peripheral adhesion complexes was detected in Palm-PTK6-YF- or Palm-PTK6-WT-expressing cells. PC3 cells were stained with anti-phosphotyrosine antibodies and visualized with FITC, which labels the peripheral adhesion complexes. Cells were counterstained with DAPI (nuclei). The size bar denotes 50 μm.

p130CAS and ERK5 are crucial for forming peripheral adhesion complexes. A, p130CAS protein is knocked down by siRNAs at day 3 and re-expressed at day 6. PC3 cells were transfected with p130CAS siRNAs or control siRNAs and harvested at 3 or 6 days. Total cell lysates were analyzed by immunoblotting with anti-p130CAS, Myc tag (PTK6), and β-catenin antibodies. B, Palm-PTK6-YF-induced peripheral adhesion complexes are disrupted upon p130CAS knockdown (si-CAS) and reassembled once p130CAS is re-expressed at day 6. Phase-contrast images and indirect immunofluorescence using anti-phosphotyrosine (PY) antibodies are shown. Size bar denotes 50 μm. C, ERK5 protein is knocked down by two ERK5 siRNAs. PC3 cells were transfected with ERK5 siRNAs (si-E5) or control siRNAs (si-Cont) and harvested at 3 days. Total cell lysates were analyzed by immunoblotting with anti-ERK5, phospho-ERK5, Myc tag, and β-catenin antibodies. D, peripheral adhesion complexes are disrupted upon ERK5 knockdown. Phase-contrast images and indirect immunofluorescence using anti-phosphotyrosine antibodies are shown. Size bars denote 50 μm. Vec, vector.

p130CAS mediates oncogenic signaling induced by Palm-PTK6-YF. A, increased p130CAS tyrosine phosphorylation and ERK5 and AKT activation were detected in Palm-PTK6-YF-expressing PC3 cells following FBS stimulation. PC3 cells stably expressing PTK6-Palm-YF or vector (Vec) were serum-starved for 48 h and stimulated with 20% FBS for 10, 30, or 60 min. Immunoblotting was performed with anti-p130CAS, phospho-p130CAS (PY-165), ERK5, phospho-ERK5, ERK1/2, phospho-ERK1/2, AKT, phospho-AKT (Thr-308), PTK6, phospho-PTK6 (PY-342), and β-catenin antibodies. B, increased ERK5 activation correlates with decreased ERK1/2 activation upon FBS stimulation in Palm-PTK6-YF-expressing cells. Relative band densities from A were quantified with NIH ImageJ software (). The density of phospho-ERK5 and phospho-ERK1/2 was normalized by the density of total ERK5 and total ERK1/2, respectively. C, increased ERK5 activation in Palm-PTK6-YF-expressing PC3 cells upon FBS stimulation was detected by indirect immunofluorescence. PC3 cells expressing vector or Palm-PTK6-YF were serum-starved for 48 h and stimulated with 20% FBS for 10 min. Phospho-ERK1/2 immunoreactivity was visualized with FITC (green), whereas phospho-ERK5 was detected with rhodamine (red). Cells were counterstained with DAPI (blue). The size bar denotes 50 μm. D, ERK5 and AKT activation was diminished upon knockdown of p130CAS. PTK6-Palm-YF-expressing PC3 cells were transfected with p130CAS siRNAs (si-CAS) or control siRNAs (si-Cont) for 24 h, then-serum starved for 48 h, and stimulated by 20% FBS for 10, 30, or 60 min. Total cell lysates were analyzed by immunoblotting with anti-p130CAS, ERK5, phospho-ERK5, AKT, phospho-AKT (Thr-308 and Ser-473), β-catenin, and α-tubulin antibodies.

Membrane-targeted PTK6 induces formation of peripheral adhesion complexes. A, increased PTK6 mRNA expression was detected in human metastatic prostate cancer samples by analyzing the NCBI human genome microarray data set GDS2545 (*, p < 0.05; **, p < 0.01). Error bars represent standard error of the mean. B, intracellular localization of PTK6 was examined in prostate cells (PC3, LNCaP, and BPH1) and breast cancer cells (MDA-MB-231 and T47D). Cells were fractionated into cytoplasmic (C), membrane/organelle (M), and nuclear (N) compartments. AKT, HER2, and SP1 were used as loading controls for cytoplasmic, membrane/organelle, nuclear compartments, respectively. C, the membrane pool of PTK6 is the active pool. Total cytoplasmic, membrane/organelle, and nuclear PC3 cell lysates of PC3 cells were subjected to immunoblotting with PTK6 and phospho-PTK6 (PY-342) antibodies. D, immunoblot analysis of total cell lysates of PC3 cells stably expressing vector (Vec), PTK6-YF, NLS-PTK-YF, or Palm-PTK6-YF was performed using anti-Myc tag (PTK6) and -α-tubulin antibodies. E, intracellular localization of exogenous PTK6 was examined by indirect immunofluorescence with anti-Myc tag antibody. Myc-tagged PTK6 immunoreactivity was visualized with FITC (green). Cells were counterstained with DAPI (blue). The size bar denotes 20 μm. F, peripheral adhesion complexes were induced in Palm-PTK6-YF-expressing cells. Phase-contrast images of PC3 cells stably expressing vector, PTK6-YF, NLS-PTK-YF, or Palm-PTK6-YF are shown. The size bar denotes 50 μm.

PTK6 directly phosphorylates p130CAS. A, increased tyrosine phosphorylation of a 130-kDa protein was observed in the presence of Palm-PTK6-YF. PC3 cells stably expressing Palm-PTK6-YF or vector (Vec) were grown in normal serum (NS), serum-starved for 48 h (SS), or serum-starved and then stimulated with 20% FBS for an hour (SS+FBS). Lysates were analyzed by immunoblotting with anti-phosphotyrosine (PY), Myc tag, and p130CAS antibodies. The black arrowhead points at a 130-kDa band, and the white arrowhead points at a 50-kDa band (phospho-Palm-PTK6). B, PTK6 directly phosphorylates p130CAS in an in vitro kinase assay. Recombinant human PTK6 and human p130CAS were incubated in kinase buffer with or without ATP for 10 min at 30 °C. Immunoblot analysis was performed using anti-phosphotyrosine, p130CAS, and PTK6 antibodies. The black arrowhead points at phospho-p130CAS, and the white arrowhead points at phospho-PTK6. C, immunoblot analysis of an in vitro kinase assay as described in B was performed using anti-phosphotyrosine (PY), phospho-p130CAS (PY-165), phospho-p130CAS (PY-664), p130CAS, and PTK6 antibodies. D, tyrosine phosphorylation of both endogenous p130CAS and exogenous GST-p130CAS was induced in the presence of active PTK6 in HEK-293 cells. p130CAS was immunoprecipitated from lysates of HEK-293 cells co-expressing GST-p130CAS and PTK6-YF. p130CAS tyrosine phosphorylation was analyzed by immunoblotting with anti-phosphotyrosine and p130CAS antibodies. The black arrowhead points at exogenous GST-p130CAS, and the white arrowhead points at endogenous p130CAS. E, PTK6 interacts with p130CAS through its SH2 domain. Glutathione-Sepharose CL-4B beads binding with GST fusion proteins were used to pull down p130CAS from HEK-293 cell lysates. Bound p130CAS was analyzed by immunoblotting with anti-p130CAS antibody. 10% of the lysates added to the pulldown reaction served as input. F, interaction between p130CAS and PTK6 depends on tyrosine phosphorylation of p130CAS. PTK6 was immunoprecipitated from lysates of SYF cells expressing vector, PTK6-YF, PTK6-KM, or PTK6-WT. p130CAS association was analyzed by immunoblotting with anti-p130CAS and PTK6 antibodies. The black arrowhead points at the band of IgG heavy chain, and the white arrowhead points at immunoprecipitated PTK6. SYF cell lysates were analyzed by immunoblotting with anti-p130CAS, phospho-p130CAS (PY-165), and PTK6 antibodies as input. IP, immunoprecipitation.

PTK6 promotes cell migration through p130CAS and ERK5 signaling. A, PC3 cell migration is diminished upon PTK6 knockdown in Transwell chamber assays using 20% FBS as a chemoattractant. B, knockdown of PTK6 by siRNA in PC3 cells lasts 4 days. Cells were transfected with control siRNA (si-Cont) or PTK6 siRNA for 24 h and harvested at 2 (d2) or 4 (d4) days after transfection. Total cell lysates were analyzed by immunoblotting with anti-PTK6, p130CAS, phospho-p130CAS (PY-165), ERK5, P-ERK5, and β-actin antibodies. C, knockdown of PTK6 results in decreased ERK5 phosphorylation in PC3 cells. Relative band densities from B were quantified with NIH ImageJ software (). Data were collected from two individual experiments, and S.D. is shown. D, PC3 cells stably expressing Palm-PTK6-YF show increased migration in wound healing assays. Representative images at 0 and 48 h are shown. Size bars denote 50 μm. E, membrane-targeted PTK6 promotes cell migration through phosphorylation of p130CAS and activation of ERK5. PC3 cells stably expressing Palm-PTK6-YF show greater cell migratory ability than control cells in Transwell chamber assays using 20% FBS as a chemoattractant. Knockdown of FAK causes a modest decrease, whereas knockdown of p130CAS or ERK5 (E5) results in a substantial reduction of cell migration induced by Palm-PTK6-YF. Error bars in A, C, and E represent standard deviation. F, a proposed model shows how PTK6 regulates peripheral adhesion complex formation and promotes migration. Integrin and growth factor receptor receptors are upstream of PTK6. PTK6 phosphorylates p130CAS near the plasma membrane (PM) and then activates ERK5 signaling, inducing formation of peripheral adhesion complexes and promoting migration. PTK6 has been shown to directly target AKT and promote AKT activation (). Palm-PTK6-YF-expressing PC3 cells show increased AKT activation upon FBS stimulation that can be impaired by knockdown of p130CAS. AKT was reported to promote migratory and invasive ability of squamous carcinoma cells (). Solid line arrows indicate direct regulation, whereas dotted line arrows represent proposed or indirect regulation. Vec, vector.