Breast tumor kinase BRK requires kinesin-2 subunit KAP3A in modulation of cell migration

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Breast tumor Kinase (BRK), also known as protein kinase 6 (PTK6), is a nonreceptor tyrosine kinase frequently overexpressed in human breast tumors.
• While BRK's role in signaling, proliferation, and migration is suggested, its precise intracellular functions and substrates remain incompletely understood.
• Existing known BRK substrates (Sam68, paxillin, STAT3) provide limited insight into BRK's broader cellular roles.

Purpose of the Study:

• To identify novel substrates of BRK using large-scale in vitro kinase assays.
• To characterize the functional relationship between BRK and its newly identified substrates, particularly in the context of cell migration.
• To elucidate the specific mechanisms by which BRK influences cellular processes through its substrates.

Main Methods:

• Screening of high-density protein filter arrays with active recombinant BRK to identify potential substrates.
• Validation of identified targets using secondary assays to confirm kinase-substrate interactions.
• In vivo characterization of kinesin associated protein 3A (KAP3A) as a BRK substrate in breast cancer cells, including phosphorylation site analysis and subcellular localization studies.

Main Results:

• Identification of at least four novel BRK targets: alpha-subunit of stimulatory guanine nucleotide binding protein (GNAS), FL139441, beta-tubulin, and kinesin associated protein 3A (KAP3A).
• Validation of KAP3A as an in vivo substrate of BRK, with BRK specifically phosphorylating C-terminal tyrosine residues.
• BRK-mediated phosphorylation of KAP3A induces its delocalization from a punctate nuclear pattern to a diffuse nucleo-cytoplasmic distribution, impacting cell migration.

Conclusions:

• Kinesin associated protein 3A (KAP3A) is identified as a novel physiological substrate of BRK.
• BRK-induced phosphorylation and subsequent delocalization of KAP3A play a crucial role in promoting breast cancer cell migration.
• KAP3A knockdown and dominant-negative mutants suppress BRK-induced cell migration, highlighting KAP3A's significance in BRK-driven oncogenic processes.