Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Abstract

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently overexpressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to Crm-1/exportin-1 mediated nuclear export. In addition, overexpression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.

Figure 1

Knockdown of cytoplasmic PTK6 reduces PC3 cell growth. (A) Immunoblot showing expression of endogenous PTK6 in stable cells containing vector (V), but great reduction of PTK6 protein expression in cells expressing shRNA targeting PTK6 (49, 52). Actin was used as a loading control. (B) Reduced growth, resulting in the shRNA expressing cell lines 49 (dashed line, open triangle) and 52 (dashed line, open circle) compared to vector (solid line, black square). (C) The average doubling time of PTK6 shRNA-expressing cells (49, 52) is increased compared to vector (V). Asterisks correspond to a p-value of <0.002 (*) and ≤0.0001 (**). (D and E) Colony formation is decreased in cells expressing PTK6 shRNA (49, 52) compared to vector (V) control. Asterisks correspond to a p-value of ≤0.001 (*) and <0.003 (**).

Figure 2

Targeting PTK6 to the nucleus of PC3 cells has a negative effect on growth. (A) Stable tetracycline-inducible PC3 cells expressing vector, wild-type nuclear-targeted (NLS-PTK6-WT) or constitutively active nuclear-targeted (NLS-PTK6-YF) PTK6 shows a slight increase in tyrosine phosphorylation upon tetracycline treatment for NLS-PTK6-WT (black arrow). NLS-PTK6-YF shows an increased tyrosine phosphorylation of several bands (black and white arrows). Myc-tagged PTK6 shows the expression level of ectopic protein. Size markers (in kDa) indicating molecular mass are shown at the left of the blot. (B) Immunofluorescence of tetracycline induced ectopic PTK6 shows expression of NLS-PTK6-WT to nuclear dots, while NLS-PTK6-YF localizes to nuclear dots and the nucleoplasm. Myc-tag staining for uninduced stable cells was used as a negative control. (C) Vector and NLS-PTK6-WT PC3 cells show no difference in growth between tetracycline induced (solid line) compared to uninduced (dotted line) conditions. NLS-PTK6-YF shows a significant decrease is cell growth for induced (solid line) compared to uninduced (dotted line) cells. (D) The average doubling time of NLS-PTK6-YF induced cells (gray) is increased compared to uninduced cells (white) and vector or NLS-PTK6-WT. Asterisks correspond to a p-value of <0.0008 (*) or <0.0003 (**). (E and F) Colony formation following tetracycline-induction of NLS-PTK6-YF (+) is greatly impaired compared with uninduced cells (−) and vector control or NLS-PTK6-WT cells. Bars are gray for tetracycline-induced conditions and white for uninduced conditions. Asterisks correspond to a p-value of <0.003 (*) or ≤0.002 (**).

Figure 3

Targeting PTK6 to the nucleus results in enhanced phosphorylation of its nuclear substrate Sam68. (A) Immunoblot of HEK293 cells transfected with GFP-Sam68 and vector, constitutively active untargeted (YF), nuclear-targeted (NLS-PTK6-YF) or membrane-targeted palmitoylated (Palm-PTK6-YF). Total cell lysate (TCL) was probed for tyrosine phosphorylation, Sam68 and Myc-epitope tagged PTK6. Ectopic GFP-Sam68 is marked with a solid arrow, endogenous Sam68 with a white arrow and ectopic Myc-tagged PTK6 with a white diamond. Size markers (in kDa) indicating molecular mass are shown at the right of the blot. (B) Co-immunoprecipitation of Sam68, probed for tyrosine phosphorylation, Sam68 and ectopic Myc-epitope tagged PTK6. Total levels of co-immunoprecipitated Sam68 and PTK6 proteins are shown in the lower panels; ectopic GFP-Sam68 (black arrow), endogenous Sam68 (white arrow) and PTK6 pulled down with Sam68 (white diamond). SE = short exposure; LE = long exposure. (C) Tyrosine phosphorylated protein compared to corresponding total protein co-immunoprecipitated was quantified using ImageJ. Tyrosine phosphorylated proteins were normalized to the corresponding total proteins pulled down.

Figure 4

Mechanisms regulating PTK6 nuclear localization are intact in PC3 cells. (A) Wild type (WT) but not constitutively activated (YF) or kinase dead (KM) PTK6 is strongly tyrosine phosphorylated in PC3 total cell lysates (TCL) transiently transfected with PTK6. (B) Immunoprecipitation of ectopic PTK6 in PC3 cells and immunoblot using anti-phosphotyrosine antibody shows differences in associations between WT, YF or KM PTK6 and tyrosine-phosphorylated proteins. The tyrosine-phosphorylated band at 50 kDa (black arrow) corresponds to Myc-epitope tagged PTK6 (white arrow). Key co-immunoprecipitated bands are marked with an open arrow head (<). Background bands corresponding to IgG are marked with an asterisk (*). (C) Fractionation of PC3 cells transfected with vector control (V) shows that endogenous PTK6 is in the cytoplasmic/membrane (C/M) fraction, but does not enter the nucleus (N). Overexpressed WT, YF and KM PTK6 are present in both the C/M and N fractions. (D) Immunofluorescence of endogenous and transiently transfected PTK6 (green) shows that endogenous protein is localized to the cytoplasm, while ectopic protein is both nuclear and cytoplasmic. Nuclei are visualized with DAPI (blue). The size bar corresponds to 20 µm.

Figure 5

Localization of endogenous PTK6 in PC3 cells is not affected by inhibition of Crm 1/exportin-1 mediated nuclear export or overexpression of its nuclear substrate Sam68. (A) Endogenous PTK6 (red) localization was not altered with leptomycin B (LMB) treatment (top right part) compared to untreated cells (top left part). GFP-tagged nuclear export signal of PKI, NES-PKI, (green) was used as a control for LMB treatment; NES-PKI is present in the nucleus of treated cells (bottom right part) compared to untreated cells (bottom left part). (B) Ectopic expression of the PTK6 substrate Sam68 (green) localizes to the nucleus, while endogenous PTK6 (red) localization remains in the cytoplasmic compartment. Nuclei are visualized with DAPI (blue). The size bar corresponds to 20 µm.

Figure 6

Inducible ectopic wild type PTK6 is retained in the cytoplasm of PC3 cells. (A) Tetracycline-induced Myc-epitope tagged wild type PTK6 was localized by immunofluorescence staining (FITC, green) and it is cytoplasmic. Nuclei are visualized with DAPI (blue). The size bar corresponds to 20 µm. (B) Fractionation of tetracycline treated PC3 cells shows that ectopically expressed Myc-epitope tagged wild type (WT) PTK6 localizes to the cytoplasmic compartment, compared to stable control cells containing empty vector (V). Tubulin, ErbB2 and Sp1 serve as markers for the cytoplasmic (C), membrane (M) and nuclear (N) fractions respectively.