The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.
RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.
Borrello
M G
MG
Divisione di Oncologia Sperimentale A, Istituto Nazionale Tumori, Milan, Italy.
Alberti
L
L
Arighi
E
E
Bongarzone
I
I
Battistini
C
C
Bardelli
A
A
Pasini
B
B
Piutti
C
C
Rizzetti
M G
MG
Mondellini
P
P
Radice
M T
MT
Pierotti
M A
MA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D029721:Drosophila Proteins; D005982:Glutathione Transferase / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D010649:Phenylalanine; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D011519:Proto-Oncogenes; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine
D029721:Drosophila Proteins; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D015513:Oncogene Proteins; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; D010738:Type C Phospholipases