Protein tyrosine kinase 6 protects cells from anoikis by directly phosphorylating focal adhesion kinase and activating AKT

Abstract

Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase expressed in epithelial cancers. Disruption of Ptk6 decreases azoxymethane-induced colon tumorigenesis in mice by preventing signal transducer and activator of transcription 3 activation. Relocalization of PTK6 in prostate cancers contributes to increased growth. Although not expressed in normal breast or ovary, PTK6 promotes anchorage-independent survival of breast and ovarian tumor cells. We identified several potential PTK6 substrates in the human SW620 colon cancer cell line using mass spectrometry, including FAK (focal adhesion kinase). We show that FAK is a direct substrate of PTK6 in vitro and in vivo. Expression of membrane-targeted active PTK6 (Palm-PTK6-YF) induces constitutive activation of FAK and cell morphology changes, which are independent of SRC family kinases in Src-/-, Yes-/-, Fyn-/- (SYF) mouse embryonic fibroblasts (MEFs). Palm-PTK6-YF expressing SYF cells are transformed and overcome contact inhibition, form colonies in transformation assays, proliferate in suspension and form tumors in a xenograft model. Expression of FAK and Palm-PTK6-YF in Fak-/- MEFs synergistically activates AKT and protects cells against anoikis. However, expression of Palm-PTK6-YF in Akt1/2-/- MEFs fails to protect cells from anoikis, indicating AKT is critical in PTK6 and FAK-mediated survival signaling. In a conditional Pten knockout murine prostate cancer model, we identify prostate epithelial cells with enhanced activation of endogenous PTK6 and FAK at the plasma membrane. Knockdown of PTK6 in the PC3 human prostate cancer cell line disrupts FAK and AKT activation and promotes anoikis, which can be rescued by exogenous expression of FAK. Our data reveal important roles for a PTK6-FAK-AKT signaling axis in promoting anchorage-independent cell survival.

Figure 1. PTK6 phosphorylates FAK independent of SRC family kinase

A. FAK is a direct PTK6 substrate. Tyrosine phosphorylation of FAK is induced by active PTK6 in the presence of ATP in an in vitro kinase assay. Immunoblotting analysis of kinase reactions was performed using anti-PY, FAK and PTK6 antibodies. B. PTK6 phosphorylates FAK at tyrosine residue 861. The LC/MS/MS tandem mass spectrum of the triply charged tryptic peptide GSIDREDGSLQ_deamGPIGNQHIpYQ_deamPVGKPDPAAPPK acquired via CID using a hybrid linear ion trap-orbitrap mass spectrometer show the phosphorylation site Y861. C. PTK6 phosphorylates FAK in cells. HEK293 and SW620 cells were transiently transfected with PCDNA3-PTK6-YF or empty vector, and total cell lysates were harvested 24 hour after transfection. HA-tag-IP or FAK-IP was performed and tyrosine phosphorylation of FAK was detected by immunoblotting with anti-PY antibodies. Immunoblotting analysis of total cell lysates was performed using anti-FAK and PTK6 antibodies as input. D. PTK6 interacts with FAK through its SH2 domain. Glutathione-Sepharose CL-4B beads binding with GST fusion proteins were used to pull down FAK from HEK-293 cell lysates. Bound FAK was analyzed by immunoblotting with anti-FAK antibodies. 10% of the lysates added to the pulldown reaction served as input. E. PTK6 induces tyrosine phosphorylation of FAK independent of SRC family kinases. SYF cells stably expressing vector, PTK6-WT, PTK6-KM or PTK6-YF were serum starved for 48 hours and stimulated by 20% FBS for 10 or 30 minutes. FAK IP was performed and tyrosine phosphorylation of FAK was detected by immunoblotting with anti-PY antibodies. Immunoblotting analysis of total cell lysates was performed using anti-FAK, P-FAK (Tyr576/Tyr577), P-FAK (Tyr925), PTK6, P-PTK6 (Tyr-342), and β-catenin antibodies. F. PTK6 induced tyrosine phosphorylation of FAK is kinase dependent. The relative band density of PY blot in the FAK IP was normalized by the density of FAK using NIH ImageJ software (50).

Figure 2. Membrane associated active PTK6 induces constitutive activation of FAK in SYF cells

A. Membrane targeted PTK6 (Palm-PTK6-YF) is constitutively active. SYF cells stably expressing vector, PTK6-YF or Palm-PTK6-YF were serum starved for 48 hours and stimulated by 20% FBS for 10 or 30 minutes. Immunoblotting analysis of total cell lysates was performed using anti-FAK, P-FAK (Tyr576/Tyr577), P-FAK (Tyr925), PTK6, P-PTK6 (Tyr342), and β-catenin antibodies. Both long (LE) and short exposures (SE) of P-FAK (Tyr925) blots are shown; the specific P-FAK band migrates at 130 kDa. B. Membrane targeted PTK6 dramatically changes the morphology of SYF cells. SYF cells expressing vector, PTK6-YF or Palm-PTK6-YF were grown at 8-well chamber slides, and indirect immunofluorescence was performed using anti-Myc-tag, P-FAK (Tyr576/Tyr577), PY and P-Paxillin (Tyr118) antibodies. Rhodamine-conjugated phalloidin was used to selectively stain F-actin. Cells were counterstained with DAPI (blue). The size bar denotes 20 μm.

Figure 3. Membrane associated active PTK6 transforms SYF MEFs

A. SYF cells stably expressing Palm-PTK6-YF are able to overcome contact inhibition. SYF cells stably expressing vector, PTK6-YF or Palm-PTK6-YF were seeded at complete growth medium, and images were taken 5 days after confluence. B. Growth curve of SYF cells as measured by CellTiter-Glo luminescent cell viability assay demonstrates the ability of Palm-PTK6-YF expressing cells to continue growing after confluence. Y axis is the fold change of relative luminescence unit (RLU), with day 1 set to 1. C. SYF cells expressing PTK6-YF or Palm-PTK6-YF show increased anchorage independent growth in soft agar assay. D. A bar graph illustrates the number of colonies formed inn soft agar assay in C (**, p < 0.01; ***, p < 0.001). E. Transformation (foci-forming) assays show increased foci-forming ability of SYF cells expressing Palm-PTK6-YF. F. Palm-PTK6-YF expressing SYF cells are able to proliferate under suspended growth conditions. Anoikis assays were performed on poly-HEMA coated plates for 6 days. G. Palm-PTK6-YF activates FAK and AKT survival signaling pathways. Palm-PTK6-YF expressing SYF cells were seeded on poly-HEMA coated plates for 72 hours. Immunoblotting analysis was performed with anti-P-PTK6 (Tyr342), PTK6, P-FAK (Tyr576/Tyr577), P-FAK (Tyr925), FAK, P-AKT (Thr308), P-AKT (Ser473), AKT, cleaved caspase 3 (CC3) and p27 antibodies. H. SYF cells expressing Palm-PTK6-YF form tumors in xenograft mice. Palm-PTK6-YF expressing cells were injected subcutaneously in left flanks of nude mice, and control cells were injected in right flanks. Tumors were indicated by white arrows. Mice were sacrificed 4 weeks after injection, and tumors were taken out for further analysis.

Figure 4. Membrane associated active PTK6 mediates protection against anoikis through activation of FAK and AKT

A. Immunoblotting analysis of lysates of Fak−/− MEFs and Akt1/2−/− MEFs demonstrates the absence of FAK in Fak−/− MEFs, and no expression of AKT in Akt1/2−/− MEFs. B. Expression of FAK and Palm-PTK6-YF (PYF) in Fak−/− MEFs shows synergistic protection against anoikis. Fak−/− MEFs stably expressing vector, FAK, Palm-PTK6-YF, or both FAK and Palm-PTK6-YF were seeded on poly-HEMA plates for 6 days. The size bar denotes 200 μm. C. Anoikis assays of Fak−/− MEFs were performed for 5 days and the cell viability was measured by CellTiter-Glo luminescent assay (**, p < 0.01; ***, p < 0.001). D. Expression of FAK and Palm-PTK6-YF synergistically activate AKT survival signaling in Fak−/− MEFs. Fak−/− MEFs stably expressing vector, FAK, Palm-PTK6-YF, or both FAK and Palm-PTK6-YF were incubated on poly-HEMA coated plates for 72 hours. Immunoblotting analysis was performed with anti-P-FAK (Tyr576/Tyr577), HA-tag, PTK6, P-AKT (Thr308), P-AKT (Ser473), AKT, cleaved caspase 3 (CC3), p27 and β-actin antibodies. E. Expression of Palm-PTK6-YF (PYF) in Akt1/2−/− MEFs fails to protect cells against anoikis. The viability of Akt1/2−/− MEFs was measured for 7 days by CellTiter-Glo luminescent assay. F. Palm-PTK6-YF induced FAK activation in Akt1/2−/− MEFs fails to protect cells against anoikis. Akt1/2−/− MEFs expressing vector or Palm-PTK6-YF were incubated on plastic plates or poly-HEMA coated plates for 24 hours. Immunoblotting analysis was performed with anti-P-FAK (Tyr576/Tyr577), P-FAK (Tyr925), FAK, P-PTK6 (Tyr342), PTK6, cleaved caspase 3 (CC3), p27 and β-actin antibodies.

Figure 5. Knockdown of PTK6 in prostate cancer cells promotes anoikis

A. A group of prostate tumor cells have highly activated PTK6 and FAK at plasma membrane in a murine prostate tumor model (PTEN^flox/flox, PB-Cre). Immunohistochemistry was performed with anti-P-PTK6 (Tyr342) and P-FAK (Tyr576/Tyr577) antibodies. The size bar denotes 20 μm. B. Knockdown of PTK6 in PC3 cells impairs FAK and AKT survival signaling under suspended condition. PC3 cells were transfected with siRNAs against PTK6 or control siRNAs for 24 hours and then seeded on plastic plates or poly-HEMA coated plates for 24 hours. Immunoblotting analysis was performed with anti-PTK6, P-FAK (Tyr576/Tyr577), P-FAK (Tyr925), FAK, P-AKT (Ser473), P-AKT (Thr308), AKT, cleaved caspase 3 (CC3), p27 and β-catenin antibodies. C. Knockdown of PTK6 promotes PC3 cells to undergo anoikis. PC3 cells transfected with siRNAs against PTK6 or control were seeded on poly-HEMA coated plates for indicated periods. Cell viability was measured by CellTiter-Glo luminescent assays. D–E. Expression of FAK is able to rescue PTK6-knockdown induced anoikis. PC3 cells were transfected with PCDNA3-HA-FAK or control plasmids for 24 hours, and then transfected with PTK6 siRNAs or control siRNAs for 24 hours. Cells were trypsinized and seeded on poly-HEMA coated plates (day 1). Protein lysates were harvested at day 2, and anoikis assays were performed at day 4 and day 6.

Fig. 6. PTK6/FAK signaling is important in regulating anoikis resistance of PC3 cells

A–B. SRC and FAK are efficiently knocked down by siRNAs in PC3 cells. PC3 cells were transfected with siRNAs against PTK6, SRC, FAK or control siRNAs for 24 hours and then seeded on poly-HEMA coated plates (day 1). Protein lysates were harvested at day 2, and immunoblotting was performed with anti-PTK6, SRC, FAK and β-catenin antibodies. Expression of β-catenin serves as a loading control. C. PTK6 is primarily responsible for anoikis resistance of PC3 cells. Anoikis assays were performed using various knockdown cells described in A-B after 120 hours of suspended growth on poly-HEMA coated plates (day 6). D. A proposed model shows how PTK6 regulates cell survival. When cells detached from extracellular matrix (ECM), membrane associated active PTK6 is able to phosphorylate and activate FAK and following AKT survival signaling. PTK6 is also able to protect cells from anoikis independent of FAK, probably through direct phosphorylation and activation of AKT (13).