Tyrosine phosphorylation of sam68 by breast tumor kinase regulates intranuclear localization and cell cycle progression.
The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.
Lukong
Kiven Erique
KE
Terry Fox Molecular Oncology Group and Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research and Department of Oncology, McGill University, Montreal, Quebec H3T 1E2, Canada.
Larocque
Daniel
D
Tyner
Angela L
AL
Richard
Stéphane
S
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology; D001921:Brain / Q000378:metabolism; D001943:Breast Neoplasms / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry*; D018450:Disease Progression; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism*; D009369:Neoplasms / Q000473:pathology*; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000737:chemistry*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489559:KHDRBS1 protein, human; D009363:Neoplasm Proteins; D010455:Peptides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; C088375:PTK6 protein, human; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516610:Ptpn1 protein, rat