Protein Tyrosine Kinase-6 (PTK6)
The intracellular protein tyrosine kinase, PTK6 (also known as the breast tumor kinase, Brk), has been implicated in the development and progression of a number of different tumor types. First identified in three separate studies in the early 1990s, it was initially found in a study to determine which tyrosine kinases were expressed in human melanocytes and then the full-length sequence was subsequently cloned from metastatic breast cancer using a screen to identify novel kinases that were expressed in tumors, but not normal breast tissue. Identification of the murine ortholog, sik, in mouse intestinal cells was achieved by the generation of a library of kinase catalytic domains.
Under normal physiological conditions, PTK6 expression is highly controlled and is usually limited to differentiating cells of epithelial origin. Several studies have reported that, pathologically (i.e., in disease tissue), PTK6 expression is increased in a number of different cancers compared to normal cells of the same tissue type (reviewed in Harvey and Burmi 2011).
The apparent disparity in PTK6 expression and the difference in proliferation status between cellular differentiation in normal tissues, where cells tend not be dividing; and the fact that tumors are highly proliferative (and cells are dividing rapidly) makes PTK6 an intriguing molecule for study, as there are likely to be both context- and function-specific differences that could be exploited in the development of antitumor therapies conferring benefit to patients.
PTK6 has been reported to have different functions in different tissue types; for example, in normal tissues PTK6’s role appears to be related to regulating the differentiation process, whereas in tumors PTK6 promotes proliferation and cell survival. Variations in cellular localization are thought to be associated with PTK6’s opposing roles in both differentiation and proliferation. Altered cellular localization will no doubt affect the variety of substrates and binding partners that are available for PTK6, thereby contributing to the different functions/effects that have been ascribed to PTK6 expression.
Myristoylation is a post- or cotranslational protein modification, whereby a fatty acid-derived group is attached to an N-terminal amino acid. Such modifications allow proteins to associate with membrane structures. Although PTK6 is structurally related to Src, it lacks the amino-terminal myristoylation site (Mitchell et al. 1994). Without a myristoylation site, PTK6 is not able to associate directly with the plasma membrane, and therefore cellular localization is not tightly regulated. Originally PTK6 was thought to be solely a cytoplasmic kinase; however, it is now known to be in different cellular compartments, including at the membrane via association with its binding partners as well as in the nucleus. Association of a protein to the plasma membrane can be mimicked by experimental inclusion of a myristoylation site. Adding a myristoylation site to the N-terminus, enhanced PTK6’s oncogenic role by promoting cell proliferation, survival, and migration of human embryonic kidney cells. Trapping PTK6 in the nucleus with a synthetic nuclear localization signal abrogated these effects (Kim and Lee 2009); these in vitro experimental effects are supported by evidence from human tumors demonstrating that normal prostate epithelial cells and well-differentiated prostate carcinomas had nuclear PTK6, whereas as poorly differentiated prostate cancers were found to have cytoplasmic PTK6 (reviewed in Goel and Lukong 2015). Increased expression of ALT-PTK6 resulted in an increase of constitutively active PTK6 in the nuclei of prostate tumor cells, suppressing proliferation (Brauer et al. 2011). Collectively, these studies suggest that PTK6’s oncogenic role may be dependent on its cellular localization; however, as PTK6 contains neither myristoylation sites nor nuclear localization signals, it is unclear how PTK6 delocalizes from one subcellular compartment to another and precisely which cellular signals are controlling this transition.
PTK6 Substrates and Binding Partners
Summary of the localization of the major identified PTK6-interacting proteins (see Goel and Lukong (2015) for a comprehensive review of the biological outputs of these interactions). A further two substrates have been reported; however the proteins involved (which are 23 kDa and 100 kDa in size) have yet to be identified
Localization of PTK6-interacting proteins
Intracellular cytoplasmic proteins
PTK6 and Signaling Pathways
From the wide variety of substrates and interacting proteins that have already been identified (Table 1), it is clear that PTK6 plays a role in a number of different cellular processes and signaling pathways (Fig. 1). This is also apparent from the range of signaling molecules that activate PTK6, which include epidermal growth factor (EGF), heregulin (HRG), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), osteopontin (OPN), and calcium ions.
PTK6 has been shown to associate with all members of the ErbB receptor family (reviewed in Hussain and Harvey 2014) and potentiates the proliferative effects of EGF by activating the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway. PTK6 is known to play a role in cell migration through the phosphorylation of paxillin, which is a ‘molecular scaffold’ that interacts with signaling molecules and a number of proteins that are involved in cell motility. In response to EGF, activated PTK6 phosphorylates paxillin resulting in the activation of Rac1 and an increase in cell migration. Treatment of breast cancer cells with the ErbB ligand HRG also increased cell migration, via activation of both Erk5 and an Erb-PTK6-Rac-p38 MAPK signaling pathway (reviewed in Goel and Lukong 2015; Ostrander et al. 2007).
PTK6’s interactions with the nuclear STAR (Signal Transduction and Activation of RNA) proteins (Sam68 and SLMs) and polypyrimidine tract-binding protein-associated splicing factor (PSF) are induced in response to EGF treatment. Phosphorylation of PSF and Sam68 (as well as the Sam68-like mammalian proteins, SLM1 and SLM2) by PTK6 results in inhibition of their RNA-binding activities. Reducing their RNA-binding capability is one mechanism of reducing the function of proteins that bind RNA. As these proteins regulate a number of RNA processing events, including alternative splicing, PTK6 activation could result in the posttranscriptional regulation of gene expression. In addition EGF-mediated activation of PTK6 induced phosphorylation of Sam68 which increased cell proliferation. This presumably occurred through suppressing the antiproliferative properties of Sam68 (Lukong et al. 2005; reviewed in Harvey and Burmi 2011).
PTK6 expression has also been linked to the potential regulation of IGF signaling. PTK6 interacts with the IGF-1R/IRS-1 complex resulting in increased activation of IGF-1R, as well as Akt. An increase in IGF-1-mediated, anchorage-independent cell survival was also observed in both breast and ovarian cancer cells (Irie et al. 2010). Similar effects on cell survival have also been shown in serum-stimulated breast cancer cells, where PTK6 again protected against cell death in suspension culture (Harvey et al. 2009). Interestingly, in both IGF-1 stimulated cells and lapatinib-resistant cells PTK6 protected cells from classical apoptosis/anoikis (Irie et al. 2010; Park et al. 2015), whereas the Harvey study showed that PTK6 protected breast cancer cells from cell death via autophagy. Taken together, these studies indicate that PTK6 can protect cells from different types of programmed cell death, and that this protection is through different mechanisms.
Furthermore, treatment of breast cancer cells with the Met receptor ligand, HGF, induced both activation of Erk5 and cell migration (Castro and Lange 2010). The PTK6/Erk5 interaction and the increased migration that was induced in response to HGF was not dependent on the kinase activity of PTK6, providing further evidence that some aspects of PTK6 function are not reliant on its kinase domain. This implies that PTK6 could coordinate the large signaling complexes that are required for cell migration, without directly phosphorylating components of the complex.
Most of the PTK6-mediated interactions and activation events result in altered cell behaviors such as increased proliferation, cell survival, migration, and the secretion of angiogenic factors, all of which are traits that are characteristic of tumor cells and are required for tumor development (reviewed in Hanahan and Weinberg 2000). It would appear from the majority of studies carried out in tumor cell lines, that PTK6 could play a central role in tumor progression, especially in breast cancer. In support of this hypothesis, Chakraborty and colleagues showed that treating cells with OPN resulted in PTK6 activation and an increase in vascular endothelial growth factor (VEGF) production. Combined with in vivo models, they were able to demonstrate that OPN triggers VEGF-dependent angiogenesis (formation of new blood vessels) and tumor growth as a result of PTK6 activation (Chakraborty et al. 2008).
In cells where PTK6 is physiologically expressed i.e., normal differentiating cells, the effects of PTK6 activation are distinct to those seen in tumor cells. When keratinocytes (skin cells) are treated with calcium, PTK6 is transiently activated and cell differentiation is induced, in contrast to the proliferation or migration seen in tumor cells. Recent evidence suggests that PTK6, the EGF receptor (EGFR), and a marker of differentiation (involucrin) may be coregulated during differentiation, and that the differentiation of normal primary human keratinocytes could be influenced by altered PTK6 expression (reviewed in Harvey and Burmi 2011; Goel and Lukong 2015).
PTK6 Expression Profile
PTK6 expression is increased in many tumors compared to normal epithelial cells (reviewed Harvey and Burmi 2011) and this is most notable and most well-characterized in breast tissue. It is only recently that PTK6 expression was detected in normal breast cells (Peng et al. 2014), with previous studies demonstrating that up to 86% of breast cancers have elevated PTK6 expression and that the increase in expression correlated with an increase in tumor grade (reviewed Harvey and Burmi 2011) is mediated by several factors including NFκB, Sp1, HIF1α, and the glucocorticoid receptor and correlates with HER2 overexpression and/or amplification in some tumors (reviewed in Goel and Lukong 2015). This is important from a tumor development standpoint, as interaction of PTK6 protein with HER2 induces and prolongs the activation of the MAPK pathway which would result in an increased cell cycle progression and tumor cell proliferation and, in mouse models, PTK6 expression promotes ErbB2-induced tumorigenesis (Peng et al. 2015).
While not the whole picture, and the clinical significance is currently far from clear, a number of studies have also reported molecular changes that could lead to elevated PTK6 expression (reviewed Harvey and Burmi 2011), regulation by microRNAs (Haines et al. 2016), as well as missense and truncating mutations that could affect PTK6 function (reviewed Goel and Lukong 2015). This suggests that there are multiple mechanisms contributing to PTK6’s overexpression.
These findings, taken together alongside the current knowledge of PTK6’s multiple signaling roles in tumor cells, suggest that PTK6 is strongly implicated in tumor development. It could therefore be expected that PTK6 expression would have a negative impact on breast cancer patient survival and Kaplan-Meier survival analysis supports this conclusion (Pires et al. 2014).
There is little doubt that the role of PTK6 is complex. It depends on a number of factors including cellular localization, accessibility of binding partners, availability of extracellular signaling molecules, and potentially, at least in tumor cells, the extent to which PTK6 expression is elevated. Further studies are fundamental to unraveling the complex and distinct roles that PTK6 plays in both normal differentiating cells and in tumor development. Progress in the isolation and development of compounds that inhibit PTK6’s kinase activity, along with recent elucidation of the crystal structure, are fundamental to furthering our understanding of the distinctions between a prodifferentiation role in normal cells, and a potentially oncogenic role in tumor progression; this is key to the development of novel therapeutic agents that could target either context-specific or cell-specific molecular functions and/or interactions with minimal effects in other tissues.
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