Breast tumor kinase and extracellular signal-regulated kinase 5 mediate Met receptor signaling to cell migration in breast cancer cells.
BACKGROUND
Breast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.


METHODS
In vitro kinase assays were performed to directly measure Brk kinase activity in response to MET and RON ligands. Transfection of Brk-targeted RNAi was used to knock down endogenous Brk or ERK5 in multiple cell lines. Kinase activities (downstream of MET signaling) were assayed by Western blotting using total and phospho-specific antibodies. Boyden chamber assays were used to measure cell migration in response to manipulation of Brk and downstream MET effectors. Rescue experiments were performed by knock down of endogenous Brk using RNAi (targeting the untranslated region (3'-UTR)) and transient transfection (re-expression) of either wild-type or kinase-inactive Brk.


RESULTS
Brk gene silencing revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk and ERK5 copurified in HGF-induced protein complexes, and Brk/ERK5 complexes formed independently of Brk kinase activity. ERK5 was required for breast cancer cell but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knockdown. Notably, rescue experiments indicated that the kinase activity of Brk was not required for HGF-induced cell migration. Further, expression of either wild-type or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration.


CONCLUSIONS
These results have identified Brk and ERK5 as important downstream effectors of Met signaling to cell migration. Targeting ERK5 kinase activity or inhibiting the formation of Brk/ERK5 complexes may provide an additional means of blocking cell migration associated with breast cancer progression to metastasis.
Castro
Nancy E
NE
Department of Pharmacology, University of Minnesota, 321 Church Street S.E., Minneapolis, MN 55455, USA.
Lange
Carol A
CA
D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005260:Female; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D048054:Mitogen-Activated Protein Kinase 7 / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D034622:RNA Interference; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; C491743:MET protein, human; D011505:Protein-Tyrosine Kinases; D019859:Proto-Oncogene Proteins c-met; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases; C088375:PTK6 protein, human; D048054:Mitogen-Activated Protein Kinase 7