Regulation of breast tumor Kinase (Brk/PTK6) downstream of Met receptor signaling leads to breast cancer cell migration.
2010-08
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Regulation of breast tumor Kinase (Brk/PTK6) downstream of Met receptor signaling leads to breast cancer cell migration.
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2010-08
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Protein tyrosine kinases (PTKs) play a critical role in the regulation of normal cell growth and differentiation and contribute to neoplastic transformation. Breast tumor kinase (Brk/PTK6) is a non-receptor or "soluble" tyrosine kinase that was cloned from a human metastatic breast tumor and found to be overexpressed in a majority (86%) of human breast tumors and cell lines. While Brk is abundant in normal differentiating epithelial cells of the GI tract and skin, it is essentially absent from normal mammary epithelial or stromal cell compartments. We hypothesize that Brk expression contributes to breast cancer progression. Previous studies in the Lange lab identified the importance of the ErbB ligand, heregulin-β1, in activating Brk kinase activity downstream of rac-1 and upstream of ERK5 and p38 MAP kinases. Stable knockdown of Brk decreased heregulin-β1-induced activation of ERK5 and p38 MAP kinases. Thus, indicating the requirement for Brk in breast cancer cell migration downstream of Her2/Her3 signaling. We sought to further probe Brk regulation as part of signaling pathways relevant to both normal and neoplastic cells. As Brk is abundantly expressed in skin, we examined Brk regulation in response to hepatocyte growth factor (HGF), and macrophage stimulating protein (MSP), peptide ligands specific for Met and Ron receptors, mediators of wound healing in skin cells and cancer cell migration during metastasis. Herein we show HGF and MSP activate Brk kinase activity in Brk+ keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells). HaCaT keratinocytes and several Brk-positive human breast cancer cell lines co-express high levels of Met and Brk mRNA and protein; HGF stimulates cell migration in these models. In vitro Brk kinase assays revealed that HGF treatment rapidly activated Brk in HaCaT, MDA-MB-231 and T47D cells, as demonstrated by increased levels of Brk autophosphorylation and phosphorylation of a recombinant Brk substrate, Sam68. Brk gene silencing studies revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk, ERK5, and Sam68 associated in HGF-induced protein complexes in both cell types; these complexes formed independently of Brk kinase activity in COS cells and independently of ERK5 kinase activity in MDA-MB-231 cells. However, ERK5 kinase activity was required for HGF-induced cell migration in MDA-MB-231 cells. ERK5 was required for breast cancer cell, but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knock-down. Notably, the protein tyrosine kinase activity of Brk was not required for HGF-induced cell migration, as indicated by rescue experiments. Further, expression of either wt or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration. Sam68 gene silencing (siRNA) experiments demonstrated that both Brk and its nuclear substrate, Sam68 are required for HGF-induced skin and breast cancer cell migration. We also identified the requirement for Sam68 phosphorylation on threonine/serine residues using a phospho-mutant Sam68 in response to HGF for increased breast cancer cell migration. Phosphorylation of Sam68 on threonine/serine residues mediates splicing of specific mRNAs. We conclude that Brk-dependent signaling to ERK5 and Sam68 mediates cell migration in response to HGF stimulation of Met receptor signaling. Met receptors are emerging as important therapeutic targets for advanced breast cancer. Targeting downstream ERK5 kinase activity or inhibiting the formation of Brk/ERK5/Sam68 complexes may provide an additional means of blocking cell migration associated with breast cancer progression towards metastasis.
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UNiversity of Minnesota Ph.D. dissertation. August 2010. Major: Pharmacology. Advisor: Carol A. Lange. 1 computer file (PDF); vii, 78 pages.
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Castro, Nancy Elizabeth. (2010). Regulation of breast tumor Kinase (Brk/PTK6) downstream of Met receptor signaling leads to breast cancer cell migration.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/95911.
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