Interleukin 2 regulates Raf-1 kinase activity through a tyrosine phosphorylation-dependent mechanism in a T-cell line.
Previously we found that interleukin 2 (IL-2) induces tyrosine phosphorylation and activation of the serine/threonine-specific kinase encoded by the raf-1 protooncogene in a T-cell line, CTLL-2. Here we extended these findings by exploring the effects of selective removal of phosphate from tyrosines in p72-74-Raf-1 kinase that had been immunoprecipitated from IL-2-stimulated CTLL-2 cells. Treatment in vitro of IL-2-activated Raf-1 with the tyrosine-specific phosphatases CD45 and TCPTP (formerly called T-cell protein tyrosine phosphatase) reduced Raf kinase activity to nearly baseline levels. This effect was completely inhibited by the phosphatase inhibitor sodium orthovanadate. In contrast, treatment of Raf-1 with a serine/threonine-specific phosphatase, protein phosphatase 1 (PP-1), resulted in a more modest decrease in Raf in vitro kinase activity, and this effect was prevented by okadaic acid. Two-dimensional phosphoamino acid analysis confirmed the selective removal of phosphate from tyrosine by CD45 and from serine and threonine by PP-1. The immunoreactivity of p72-74-Raf-1 with anti-phosphotyrosine antibodies was also completely abolished by treatment with CD45 in the absence but not in the presence of sodium orthovanadate. These findings provide evidence that the IL-2-stimulated phosphorylation of Raf-1 on tyrosines plays an important role in upregulating the activity of this serine/threonine-specific kinase in CTLL-2 cells and, as such, provides a model system for studying the transfer of growth factor-initiated signals from protein tyrosine kinases to serine/threonine-specific kinases.
Turner
B C
BC
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6082.
Tonks
N K
NK
Rapp
U R
UR
Reed
J C
JC
D000818:Animals; D002460:Cell Line; D004988:Ethers, Cyclic / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D019319:Okadaic Acid; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D019000:Phosphotyrosine; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000201:enzymology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D004988:Ethers, Cyclic; D007376:Interleukin-2; D010710:Phosphates; D011518:Proto-Oncogene Proteins; D019319:Okadaic Acid; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1