RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism.
We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.
Gorla
L
L
Proteomics Laboratory, Department of Experimental Oncology and Lab. Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan Italy.
Cantù
M
M
Miccichè
F
F
Patelli
C
C
Mondellini
P
P
Pierotti
M A
MA
Bongarzone
I
I
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D018814:Multiple Endocrine Neoplasia Type 2b / Q000235:genetics / Q000378:metabolism; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013961:Thyroid Gland / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489559:KHDRBS1 protein, human; D020033:Protein Isoforms; D011506:Proteins; D016601:RNA-Binding Proteins; D014443:Tyrosine; D012313:RNA; D051096:Proto-Oncogene Proteins c-ret