Although PTK6 shares overall structural similarity with Src, it lacks an N-terminal myristoylation/palmitoylation consensus sequence that is found in all Src family members (51
). Lack of such a membrane-targeting signal appears to facilitate its localization to different cellular compartments, including the nucleus (14
). In the prostate, PTK6 is localized to nuclei of normal luminal epithelial cells but translocated to the cytoplasm in prostate cancer (14
). These data suggested that PTK6 may play an oncogenic role when it is localized to the cytoplasm. This notion was supported by a recent report that showed that cytoplasm/membrane-targeted PTK6 promotes HEK-293 cell proliferation, cell survival, migration, and anchorage-independent growth, while nuclearly targeted PTK6 does not (25
). In addition, our group recently demonstrated that PTK6 either positively or negatively regulates β-catenin transcriptional activity depending on its intracellular localization (41
). Here we show that PTK6 preferentially phosphorylates AKT in the cytoplasm, compared with that in the membrane compartment (Fig. and ).
We demonstrate that AKT is a direct substrate of PTK6, and we have identified tyrosine residues 315 and 326 as the major sites in AKT that are phosphorylated by PTK6. It has been reported that Src also phosphorylates AKT on these two tyrosine residues (11
). However, PTK6 is able to induce tyrosine phosphorylation in the absence of Src, Yes, and Fyn kinases in SYF cells. We examined the possible significance of phosphorylation of AKT tyrosine residue 215, which was identified as a target by mass spectrometry in our studies, and tyrosine residue 474, which was phosphorylated after the treatment of tyrosine phosphatase inhibitor pervanadate (12
). However, mutation of tyrosine residues 215 and 474 to phenylalanine did not affect tyrosine phosphorylation of AKT induced by active PTK6, indicating that they are not key PTK6 target sites (data not shown). However, since tyrosine phosphorylation of AKT can still be detected after mutation of tyrosine 315 and 326 to phenylalanine (Fig. ), it is possible that other tyrosine site(s) are targeted by PTK6.
Similar to Src family kinases, the SH3 and SH2 domains of PTK6 play important roles in substrate recognition (24
). Src interacts with AKT through its SH3 domain, which recognizes the proline-rich motif (P424
) located at the AKT C terminus (27
). Although the SH3 domain of PTK6 shares only approximately 35% amino acid identity with the Src SH3 domain (3
), we found that PTK6 associates with AKT through the same proline-rich motif, P424
, in AKT. Mutation of AKT proline residues 424 and 427 impaired association between PTK6 and AKT (Fig. ). We also demonstrated that the SH2 domain of PTK6, which recognizes phosphorylated tyrosines, also plays a role in regulating PTK6-AKT interactions. Mutation of AKT tyrosine residues 315 and 326 to phenylalanine diminishes association between PTK6 and AKT (Fig. ), and kinase-active PTK6-YF promotes PTK6-AKT association (Fig. ). The tyrosine residues targeted by PTK6 are located in the catalytic domain of AKT, and regulated association of PTK6 with AKT through kinase-independent SH3- and/or kinase-dependent SH2-mediated interactions may have an impact on AKT activity.
While phosphorylation of AKT on Ser-473 and Thr-308 is critical for AKT activation, phosphorylation of AKT on tyrosine residues is also becoming recognized as important for modulation of its activities. Both positive and negative roles for tyrosine phosphorylation in regulating AKT activity have been reported (11
). We demonstrate a role for PTK6 in sensitizing SYF cells to a physiological concentration of EGF (0.1 ng/ml) leading to increased AKT activation and cell proliferation. This oncogenic role for PTK6 is kinase dependent. These experiments were performed in a background without the Src, Yes, and Fyn kinases, allowing us to delineate the specific roles of PTK6 more clearly. Our data are consistent with a previous publication that showed that phosphorylation of AKT on tyrosine residues 315 and 326 contributes to AKT activation (11
) and with the observation that overexpression of PTK6 promotes breast cancer cell growth through stimulating ErbB receptor family signaling and PI3K/AKT signaling (22
Although PTK6 appears to enhance oncogenic signaling in breast cancer cells, its activities have been correlated with differentiation and stress-induced apoptosis in normal tissues. Disruption of the Ptk6
gene led to enhanced proliferation and delayed enterocyte differentiation in the untreated mouse intestine (20
). In addition, radiation-induced apoptosis was impaired in the Ptk6
null mouse (21
). Both phenotypes were accompanied by an increase in activation of AKT signaling, leading to the hypothesis that PTK6 acts as an inhibitor of AKT in normal tissues. In addition, PTK6 was found to inhibit AKT in PTK6/AKT complexes in unstimulated cultured cells (55
). When these studies were initiated, our goal was to determine how PTK6 inhibited AKT activities in mouse tissues and cells. However, although we determined that AKT is a direct PTK6 substrate, we did not discover a kinase-dependent inhibitory role for PTK6 in AKT regulation. In contrast, we found that PTK6 may stimulate AKT activation and cell proliferation in response to physiological levels of growth factors. The underlying mechanisms for PTK6-mediated inhibition of AKT activation may involve kinase-independent association/sequestration of AKT by PTK6 that is induced during differentiation or stress. Recent studies that are under way suggest that PTK6 may also promote activation of a phosphatase that targets activating phosphorylation of AKT (A. O. Perekatt and A. L. Tyner, unpublished data).
Like several other regulatory proteins, PTK6 functions appear dependent on cellular context, coexpression of other interacting signaling molecules, and its intracellular localization. A correlation between PTK6 and ErbB2 overexpression was revealed in invasive ductal breast carcinomas (8
), and PTK6 increases the ErbB2-induced activation of Ras/MAPK signaling to induce cell proliferation in breast cancers (54
). Taken together, our studies suggest that PTK6 may also promote ErbB receptor signaling by enhancing AKT activation in response to physiological levels of growth factors. These studies provide insight about the potential benefits of targeting PTK6 as part of a therapeutic regimen to treat different types of cancer that have upregulated PTK6 expression and activity.