Interleukin 2 Regulates Raf-1 Kinase Activity Through a Tyrosine Phosphorylation-Dependent Mechanism in a T-Cell Line

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-06, Vol.90 (12), p.5544-5548
• Main Authors: Turner, Bruce C., Tonks, Nicholas K., Rapp, Ulf R., Reed, John C.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Biological and medical sciences; Cell growth; Cell Line; Cell lines; Cell physiology; Cellular metabolism; Ethers, Cyclic - pharmacology; Fundamental and applied biological sciences. Psychology; Immunity (Disease); Interleukin-2 - pharmacology; Kinetics; Mice; Molecular and cellular biology; Okadaic Acid; Phosphatases; Phosphates; Phosphates - metabolism; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphorylation; Phosphotyrosine; Protein Phosphatase 1; Protein-Serine-Threonine Kinases - metabolism; Proteins; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-raf; Signal transduction; Sodium; T lymphocytes; T-Lymphocytes, Cytotoxic - drug effects; T-Lymphocytes, Cytotoxic - enzymology; Tumors; Tyrosine - analogs & derivatives; Tyrosine - metabolism; Vanadates; Vanadates - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.12.5544

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.