Breast tumor kinase BRK requires kinesin-2 subunit KAP3A in modulation of cell migration.
BReast tumor Kinase (BRK) also known as protein kinase 6 (PTK6) is a nonreceptor tyrosine kinase overexpressed in the majority of human breast tumors. Although some studies have implicated BRK in signalling, cell proliferation and migration, the precise intracellular role of BRK has not been fully elucidated. The RNA-binding protein Sam68, and adaptor proteins paxillin and STAT3 are the only BRK substrates that link BRK to signal transduction. To identify new BRK substrates, we screened high-density protein filter arrays by large-scale in vitro kinase assays using active recombinant BRK. We identified at least 4 BRK targets comprising the alpha-subunit of stimulatory guanine nucleotide binding protein (GNAS), FL139441, beta-tubulin and kinesin associated protein 3A (KAP3A) and validated them as BRK substrates using a secondary assay. Further characterization revealed that KAP3A is an in vivo substrate of BRK and associates with BRK in breast cancer cells. We show that BRK specifically phosphorylated tyrosine residues at the C-terminus of KAP3A and induces delocalization of KAP3A from punctate nuclear localization to a diffuse nucleo-cytoplasmic pattern. Functionally, we demonstrate that KAP3A knockdown results in suppression of BRK-induced migration of breast cancer cells and show that the C-terminal deletion mutant of KAP3A acts as a dominant negative in BRK-induced cell migration. Our findings therefore reveal new substrates of BRK and define KAP3A as a physiological substrate of BRK during cell migration.
Lukong
Kiven E
KE
Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal Québec, Canada.
Richard
Stéphane
S
D000595:Amino Acid Sequence; D002384:Catalysis; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D040081:Protein Array Analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014162:Transfection
D050505:Mutant Proteins; D009363:Neoplasm Proteins; D021122:Protein Subunits; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C088375:PTK6 protein, human; C527989:KRTAP3-4P protein, human; D016547:Kinesins