Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Abstract

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10,29,30,36,40,49,54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ra...

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

“…Recently we determined that PTK6 is able to directly phosphorylate and activate cytoplasmic AKT but not membrane targeted AKT and AKT activation may enhance growth and survival of cancer cells. 35 Targeting PTK6 to the nucleus inhibited β-catenin/TCF transcription, while targeting PTK6 to the membrane activated β-catenin/TCF transcription in the SW620 colon carcinoma cell line. 50 PTK6 mediated tyrosine phosphorylation of nuclear PSF, a polypyrimidine tract-binding (PTB) protein-associated splicing factor, resulted in its cytoplasmic relocalization and cell cycle arrest, demonstrating a growth-suppressive role for PTK6 when targeting nuclear substrates.…”

PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer via SNAIL-Dependent E-Cadherin Regulation

“…PTK6 inhibitor treatment of mesenchymal TNBC cells suppressed metastasis formation, again phenocopying the effects of PTK6 shRNA expression. Although there are a growing number of PTK6 substrates, such as AKT, b-catenin, STAT3/5, Paxillin, SAM68 (29,(43)(44)(45)(46)(47)(48), none has been specifically linked to regulation of EMT in TNBC. The specific substrates of PTK6 responsible for regulation of SNAIL and E-cadherin are the focus of current, ongoing studies.…”

Protein-tyrosine Kinase 6 Promotes Peripheral Adhesion Complex Formation and Cell Migration by Phosphorylating p130 CRK-associated Substrate

“…Plasmids and siRNAs-Myc-tagged full-length human wild type (WT), active (YF), and kinase-defective (kinase-dead mutant (KM)) PTK6 in the pcDNA3 vector as well as Myctagged NLS-PTK6 and Palm-PTK6 constructs in the pcDNA4-TO vector have been described previously (9,12). Coding sequences from the pcDNA4-TO constructs were subcloned into the pBABE-puro vector (Cell Biolabs, Inc., San Diego, CA).…”

“…Coding sequences from the pcDNA4-TO constructs were subcloned into the pBABE-puro vector (Cell Biolabs, Inc., San Diego, CA). The GST-PTK6 (mouse) constructs were described previously (9). The pEBG-p130CAS plasmid (plasmid 15001; Ref.…”

“…For the GST pulldown assay, GST fusion proteins were expressed in BL21 cells (Stratagene, La Jolla, CA). GST protein purification and the subsequent processes were performed as described previously (9).…”