Tyrosine phosphorylation of GAP and GAP-associated proteins in lymphoid and fibroblast cells expressing lck.
The Ras GTPase activating protein (GAP) is a strong candidate for the protein that links protein-tyrosine kinases to the Ras mitogenic pathway. GAP and two associated proteins, p62 and p190, were shown to be phosphorylated on tyrosine in the LSTRA thymoma cell line, in which the p56lck tyrosine kinase is overexpressed as a result of retroviral promoter insertion. In NIH3T3 fibroblasts expressing specific oncogenic and transformation-defective variants of p56lck, we found that the tyrosine phosphorylation of GAP complexes required both enzymatic activation and myristylation of p56lck, and correlated with lck transforming activity. The interaction between p62 and p190 from lck-transformed fibroblasts and GAP could be reconstituted in vitro using bacterial TrpE fusion proteins containing GAP Src homology 2 (SH2) domains. In vitro complex formation was insensitive to the prior denaturation of SH2 ligands, suggesting that SH2-binding sites are formed by linear peptide sequences. These results suggest that the tyrosine phosphorylation of GAP, and its interactions with SH2-binding proteins, may be involved in fibroblast transformation by activated lck, and may participate in signal transduction and cellular transformation in lymphoid cells.
Ellis
C
C
Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.
Liu
X Q
XQ
Anderson
D
D
Abraham
N
N
Veillette
A
A
Pawson
T
T
D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013945:Thymoma / Q000378:metabolism / Q000473:pathology; D013953:Thymus Neoplasms / Q000378:metabolism / Q000473:pathology
D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019204:GTP-Binding Proteins