Abstract Protein tyrosine kinase 6 (PTK6) has context-specific, kinase-dependent and -independent functions in normal cells and cancer. In prostate cells, nuclear PTK6 suppresses growth, while active membrane-associated PTK6 promotes oncogenic signaling. Unlike SRC-family kinases, PTK6 is not modified by lipidation to promote membrane association. Although arginine residues in the PTK6 SH2 domain were previously linked to lipid binding, we found they are instead critical for protein stability and arginine methylation. Mutation of R131/R136 or R132 impaired PTK6 ubiquitination and its association with a Cullin-1-RING ubiquitin ligase complex, and its half-life increased from one hour to over 24 h. We show that PTK6 is targeted by PRMT1 and mutation of PTK6 R131/R136 or R132, or treatment with the type 1 PRMT inhibitor GSK3368715, leads to decreased arginine methylation, increased PTK6 protein stability and impaired PTK6 association with its substrates. In prostate cancer cells, ectopic expression of mutant PTK6 R131/R136 or R132 leads to reduced proliferation, anchorage-independent growth, and cell migration and invasion, when compared with wild type PTK6, even though higher levels of mutant protein are expressed. These results indicate that arginine methylation is a key regulatory switch for PTK6 signaling and protein turnover and may be central for the execution of its diverse context-specific functions. Mutation of PTK6 arginine residues has been reported in some cancers, which may contribute to disease-specific PTK6 expression and signaling.

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is distantly related to the SRC family of tyrosine kinases. It is expressed in epithelial linings and regulates regeneration and repair of the intestinal epithelium. Analysis of publicly available datasets showed Ptk6 is upregulated in tuft cells upon activation of type 2 immunity. We found that disruption of Ptk6 influences gene expression involved in intestinal immune responses. Administration of succinate, which mimics infection and activates tuft cells, revealed PTK6-dependent activation of innate immune responses in male but not female mice. In contrast to all wild type and Ptk6-/- female mice, Ptk6-/- male mice do not activate innate immunity or upregulate differentiation of the tuft and goblet secretory cell lineages following succinate treatment. Mechanistically, we found that PTK6 regulates Il25 and Irag2, genes that are required for tuft cell effector functions and activation of type 2 innate immunity, in organoids derived from intestines of male but not female mice. In patients with Crohn's disease, PTK6 is upregulated in tuft cells in noninflamed regions of intestine. These data highlight roles for PTK6 in contributing to sex differences in intestinal innate immunity and provide new insights into the regulation of IL-25.

Tuft cells are chemosensory epithelial cells that increase in number following infection or injury to robustly activate the innate immune response to alleviate or promote disease. Recent studies of castration resistant prostate cancer and its subtype, neuroendocrine prostate cancer, revealed Pou2f3+ populations in mouse models. The transcription factor Pou2f3 is a master regulator of the tuft cell lineage. We show that tuft cells are upregulated early during prostate cancer development, and their numbers increase with progression. Cancer-associated tuft cells in the mouse prostate express DCLK1, COX1, COX2, while human tuft cells express COX1. Mouse and human tuft cells exhibit strong activation of signaling pathways including EGFR and SRC-family kinases. While DCLK1 is a mouse tuft cell marker, it is not present in human prostate tuft cells. Tuft cells that appear in mouse models of prostate cancer display genotype-specific tuft cell gene expression signatures. Using bioinformatic analysis tools and publicly available datasets, we characterized prostate tuft cells in aggressive disease and highlighted differences between tuft cell populations. Our findings indicate that tuft cells contribute to the prostate cancer microenvironment and may promote development of more advanced disease. Further research is needed to understand contributions of tuft cells to prostate cancer progression.

Expression of Protein tyrosine kinase 6 (PTK6) is upregulated in several human solid tumors, and it has oncogenic roles in prostate and breast cancer. PTK6 and SRC kinase are distantly related, share many substrates, and often regulate the same signaling pathways, but whether they interact to regulate signaling is not well understood. We characterized crosstalk between PTK6 and SRC and show that PTK6 can directly phosphorylate SRC to promote its activation. Stable knockdown of PTK6 in multiple cancer cell lines leads to decreased activating phosphorylation of SRC. We show that coexpression of kinase-dead SRC and active PTK6 in mouse embryonic fibroblasts lacking Src, Yes, and Fyn results in activating phosphorylation of SRC. However, there is no reciprocal effect, because active SRC does not promote activating phosphorylation of PTK6. Overexpression of active PTK6 maintained activation of epidermal growth factor receptor (EGFR), AKT, and FAK, but not SHP2 and ERK1/2 in cells with knockdown of SRC. Both PTK6 and SRC are regulated by EGFR, and its inhibition with erlotinib downregulated PTK6 and to a lesser degree SRC activation in LNCaP cells that overexpress active PTK6. Erlotinib treatment also led to AKT inhibition, but overexpression of active PTK6 prevented this. Our data demonstrate overlapping and unique functions for PTK6 and SRC. Finally, we show that PTK6 and SRC are coexpressed in subsets of human prostate and breast cancer cells, and active PTK6 and active SRC colocalize in prostate cancer, supporting a role for PTK6 in promoting SRC activity in cancer.

Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6–vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.

PTEN activity is often lost in prostate cancer. We show that the tyrosine kinase PTK6 (BRK) is a PTEN substrate. Phosphorylation of PTK6 tyrosine 342 (PY342) promotes activation, while phosphorylation of tyrosine 447 (PY447) regulates auto-inhibition. Introduction of PTEN into a PTEN null prostate cancer cell line leads to dephosphorylation of PY342 but not PY447 and PTK6 inhibition. Conversely, PTEN knockdown promotes PTK6 activation in PTEN positive cells. Using a variety of PTEN mutant constructs, we show that protein phosphatase activity of PTEN targets PTK6, with efficiency similar to PTP1B, a phosphatase that directly dephosphorylates PTK6 Y342. Conditional disruption of Pten in the mouse prostate leads to tumorigenesis and increased phosphorylation of PTK6 Y342, and disruption of Ptk6 impairs tumorigenesis. In human prostate tumor tissue microarrays, loss of PTEN correlates with increased PTK6 PY342 and poor outcome. These data suggest PTK6 activation promotes invasive prostate cancer induced by PTEN loss.

Background: Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular SRC-related tyrosine kinase that does not possess canonical localization signals. Lack of targeting yields flexibility in its intracellular localization and access to substrates. PTK6 is observed in the nuclei of prostate epithelial cells where it promotes differentiation and inhibits growth. PTK6 is activated at the plasma membrane in PTEN-deficient mouse prostate tumors where the concentration of PIP3 is high due to PTEN loss of function. Membrane-associated PTK6 induces cell transformation, the epithelial-mesenchymal transition, and tumor cell metastasis. In human prostate cancer, active PTK6 associates with the plasma membrane and its increased expression correlates with metastasis and poor survival. Our studies aimed to characterize how the mislocalization and activation of PTK6 in prostate cancer is regulated. Results: Based on our observation that loss of PTEN induces accumulation of PTK6 at the membrane, we hypothesized that PTK6 binds to the plasma membrane secondary messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Amino acid sequence alignment predicts H52, Y53, and K54 of PTK6 as a potential PIP3 binding site, which resembles the lipid-binding HYK domain of tyrosine kinases BTK and Abl. Using NMR saturation transfer difference (STD) experiment, we observed that the SH2 domain of PTK6 contains a binding site for inositol trisphosphate (IP3) a soluble PIP3 mimic. As predicted, statistical analysis identified Y53 of the PTK6 HYK motif within the SH2 domain as a residue with substantial chemical shift perturbations (CSPs) induced by independent titrations with PIP3 analogs IP3, IP4, and 1,2-dihexanoyl PIP3 in 15N HSQC, as compared to controls. Using autophosphorylated and unphosphorylated PTK6, NMR STD experiments show that only active (pY342) PTK6 binds to IP3. Treatment of prostate cancer cells with the PI3K inhibitor wortmannin reduces active PTK6 at the plasma membrane. Conclusions: Our data demonstrate that active PTK6 is recruited to the plasma membrane through direct interactions with PIP3. Membrane association places PTK6 in close proximity to its oncogenic substrates AKT, FAK and BCAR1, thereby promoting tumor progression. These findings may facilitate identification of novel PTK6 inhibitors that block its association with the membrane, while not affecting its tumor suppressive role in the nucleus. This work is funded by NIH grant 1R01CA188427 to ALT and VG. Citation Format: Darren J. Wozniak, Ben Hitchinson, Wenjun Bie, Vadim Gaponenko, Angela L. Tyner. Phospholipids regulate the localization and oncogenic potential of protein tyrosine kinase 6 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 197.

Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and the skin, where it is expressed in nondividing differentiated cells. We found that PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6 -/- SENCAR mice to incremental doses of UVB for 30 weeks. Wild-type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of signal transducer and activator of transcription-3 (STAT3) than Ptk6-/- mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild-type mice following UVB treatment. However, wild-type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6-/- mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1). Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 has roles in normal differentiation, it also contributes to UVB-induced injury and tumorigenesis in vivo.

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial-mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426-37. ©2013 AACR.

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that has distinct functions in normal epithelia and cancer. It is expressed primarily in nondividing epithelial cells in the normal intestine, where it promotes differentiation. However, after DNA damage, PTK6 is induced in proliferating progenitor cells, where it contributes to apoptosis. We examined links between PTK6 and the tumor suppressor p53 in the isogenic p53+/+ and p53−/− HCT116 colon tumor cell lines. We found that p53 promotes expression of PTK6 in HCT116 cells, and short hairpin RNA-mediated knockdown of PTK6 leads to reduced induction of the cyclin-dependent kinase inhibitor p21. Knockdown of PTK6 enhances apoptosis in HCT116 cells with wild-type p53, following treatment of cells with γ-radiation, doxorubicin, or 5-fluorouracil. No differences in the activation of AKT, ERK1/2, or ERK5, known PTK6-regulated prosurvival signaling proteins, were detected. However, activity of STAT3, a PTK6 substrate, was impaired in cells with knockdown of PTK6 following DNA damage. In contrast to its role in the normal epithelium following DNA damage, PTK6 promotes survival of cancer cells with wild-type p53 by promoting p21 expression and STAT3 activation. Targeting PTK6 in combination with use of chemotherapeutic drugs or radiation may enhance death of colon tumor cells with wild-type p53. Mol Cancer Ther; 11(11); 2311–20. ©2012 AACR.

BACKGROUND & AIMS: Protein tyrosine kinase 6 (PTK6) is expressed throughout the gastrointestinal tract and is a negative regulator of proliferation that promotes differentiation and DNA-damage-induced apoptosis in the small intestine. PTK6 is not expressed in normal mammary gland, but is induced in most human breast tumors. Signal transducer and activator of transcription 3 (STAT3) mediates pathogenesis of colon cancer and is a substrate of PTK6. We investigated the role of PTK6 in colon tumorigenesis. METHODS: Ptk6(+/+) and Ptk6(-/-) mice were injected with azoxymethane alone or in combination with dextran sodium sulfate; formation of aberrant crypt foci and colon tumors was examined. Effects of disruption of Ptk6 on proliferation, apoptosis, and STAT3 activation were examined by immunoblot and immunohistochemical analyses. Regulation of STAT3 activation was examined in the HCT116 colon cancer cell line and young adult mouse colon cells. RESULTS: Ptk6(-/-) mice developed fewer azoxymethane-induced aberrant crypt foci and tumors. Induction of PTK6 increased apoptosis, proliferation, and STAT3 activation in Ptk6(+/+) mice injected with azoxymethane. Disruption of Ptk6 impaired STAT3 activation following azoxymethane injection, and reduced active STAT3 levels in Ptk6(-/-) tumors. Stable knockdown of PTK6 reduced basal levels of active STAT3, as well as activation of STAT3 by epidermal growth factor in HCT116 cells. Disruption of Ptk6 reduced activity of STAT3 in young adult mouse colon cells. CONCLUSIONS: PTK6 promotes STAT3 activation in the colon following injection of the carcinogen azoxymethane and regulates STAT3 activity in mouse colon tumors and in the HCT116 and young adult mouse colon cell lines. Disruption of Ptk6 decreases azoxymethane-induced colon tumorigenesis in mice.

Disruption of the gene encoding protein tyrosine kinase 6 (PTK6) leads to increased growth, impaired enterocyte differentiation and higher levels of nuclear β-catenin in the mouse small intestine. Here, we demonstrate that PTK6 associates with nuclear and cytoplasmic β-catenin and inhibits β-catenin- and T-cell factor (TCF)-mediated transcription. PTK6 directly phosphorylates β-catenin on Tyr64, Tyr142, Tyr331 and/or Tyr333, with the predominant site being Tyr64. However, mutation of these sites does not abrogate the ability of PTK6 to inhibit β-catenin transcriptional activity. Outcomes of PTK6-mediated regulation appear to be dependent on its intracellular localization. In the SW620 colorectal adenocarcinoma cell line, nuclear-targeted PTK6 negatively regulates endogenous β-catenin/TCF transcriptional activity, whereas membrane-targeted PTK6 enhances β-catenin/TCF regulated transcription. Levels of TCF4 and the transcriptional co-repressor TLE/Groucho increase in SW620 cells expressing nuclear-targeted PTK6. Knockdown of PTK6 in SW620 cells leads to increased β-catenin/TCF transcriptional activity and increased expression of β-catenin/TCF target genes Myc and Survivin. Ptk6-null BAT-GAL mice, containing a β-catenin-activated LacZ reporter transgene, have increased levels of β-galactosidase expression in the gastrointestinal tract. The ability of PTK6 to negatively regulate β-catenin/TCF transcription by modulating levels of TCF4 and TLE/Groucho could contribute to its growth-inhibitory activities in vivo.

zone, a specific PPARγ agonist, increased gastrin gene expression and Bcl-2 and PKB/Akt protein levels in each of cancer cell lines examined. Furthermore, transfection of the PANC1, HT29 and HCT116 cells with a PPARγ siRNA reversed ciglitazone-induced PKB/Akt phosphorylation and Bcl-2 protein up-regulation. Conclusions: 1) Gastrin peptide promotes cancer cells growth via an increase in annexin II cell surface concentration in these cells; 2) gastrin cooperates with the tumour-growth promoting activity of PPARγ agonists.

zone, a specific PPARγ agonist, increased gastrin gene expression and Bcl-2 and PKB/Akt protein levels in each of cancer cell lines examined. Furthermore, transfection of the PANC1, HT29 and HCT116 cells with a PPARγ siRNA reversed ciglitazone-induced PKB/Akt phosphorylation and Bcl-2 protein up-regulation. Conclusions: 1) Gastrin peptide promotes cancer cells growth via an increase in annexin II cell surface concentration in these cells; 2) gastrin cooperates with the tumour-growth promoting activity of PPARγ agonists.

BACKGROUND & AIMS: Protein tyrosine kinase 6 (PTK6) is expressed in epithelial linings of the gastrointestinal tract. PTK6 sensitizes the nontransformed Rat1a fibroblast cell line to apoptotic stimuli. The aim of this study was to determine if PTK6 regulates apoptosis in vivo after DNA damage in the small intestine. METHODS: Wild-type and Ptk6(-/-) mice were subjected to gamma-irradiation; intestinal tissues were collected, protein was isolated, and samples were fixed for immunohistochemical analyses at 0, 6, and 72 hours after the mice were irradiated. Expression of PTK6 was examined in the small intestine before and after irradiation. Apoptosis and proliferation were compared between wild-type and Ptk6(-/-) mice. Expression and activation of prosurvival signaling proteins were assessed. RESULTS: Irradiation induced PTK6 in crypt epithelial cells of the small intestine in wild-type mice. Induction of PTK6 corresponded with DNA damage-induced apoptosis in the wild-type small intestine. Following irradiation, the apoptotic response was impaired in the intestinal crypts of Ptk6(-/-) mice. Increased activation of AKT and extracellular signal-regulated kinase (ERK)1/2 and increased inhibitory phosphorylation of the proapoptotic protein BAD were detected in Ptk6(-/-) mice after irradiation. In response to the induction of apoptosis, compensatory proliferation increased in the small intestines of wild-type mice but not in Ptk6(-/-) mice at 6 hours after irradiation. CONCLUSIONS: PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting prosurvival signaling. After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of AKT and ERK1/2.

Background and Aims: PTK6 is an intracellular src-related tyrosine kinase that regulates differentiation in the intestine, where knockout animals have increased proliferative activity and growth characteristics. To explore the phenotype further we attempted to establish epithelial cell lines from the intestinal mucosa.Method: We mated Ptk6 null mice with a tsSV40 large T transgenic mouse (Immortomouse) to obtain null mice carrying the SV40 gene. Intestinal tissues from these mice were cultured.Results: We established a Ptk6 null epithelial cell line from the colonic mucosa. Consistent with a role of Ptk6 in cell differentiation, these cells have the characteristics of a stable progenitor cell. In monolayer culture, the cells form domes in the monolayer when confluent. When cultured on Transwell filters, the cells polarize and form an electrically resistant barrier. Formation of tight junctions was confirmed by demonstrating expression of ZO1 and occludin at the apical junctions, whereas E-cadherin localized to the basolateral membrane. When cultured in collagen gel, the Ptk6 null cells form complex organoids, some of which resemble cups of cells. These organoids contain cells with differentiated phenotypes. Using immunohistochemistry and confocal microscopy we have been able to identify villin-positive (absorptive cells) and a small percentage of mucin-containing cells (goblet cells) and chromogranin A-positive cells (endocrine cells).Conclusion: This conditionally immortalized cell line represents an excellent cell culture model system for exploring the mechanisms of cell function and epithelial differentiation in the colonic mucosa.