Identification of a Sam68 ribonucleoprotein complex regulated by epidermal growth factor.
Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of approximately 40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200-450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex.
Huot
Marc-Etienne
ME
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 H3T 1E2, Canada.
Vogel
Gillian
G
Richard
Stéphane
S
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D018960:Hyaluronan Receptors; C489559:KHDRBS1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D004815:Epidermal Growth Factor; D013755:Tetradecanoylphorbol Acetate