Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 Raf-1 kinase in a T-cell line.

Abstract

Interleukin 2 (IL-2) is a lymphokine, produced by T cells upon antigenic or mitogenic stimulation, that is a critical regulator of T-cell proliferation. Although the binding of IL-2 to its receptor has been well characterized, the molecular mechanisms by which IL-2 transmits its signal from the membrane to the interior of the cell are poorly understood. Like most other growth factors, IL-2 causes rapid phosphorylation of proteins within its target cells. Unlike many other growth factors, however, the known subunits of the IL-2 receptor lack tyrosine-specific kinase activity, and little is known about the kinases whose activities are regulated by IL-2.Here we show that IL-2 (but not IL-4) induces rapid phosphorylation of the p72-74 serine/threonine-specific kinase encoded by the c-Raf-1 protooncogene in an IL-2-dependent murine T-cell line, CTLL-2, and that this phosphorylation is associated with increased kinase activity in p72-74 Raf-icontaining immune complexes. The concentration dependence of IL-2-mediated elevations in Raf-1 kinase activity correlated well with IL-2-stimulated proliferation of CTLL-2 cells. Furthermore, much of the IL-2-stimulated phorphorylation of p72-74 Raf-1 occurred on tyrosines. To our knowledge, the Raf-1 kinase represents the first endogenous substrate of an IL-2-regulated tyrosine kinase to be identified.Interleukin 2 (IL-2) is thought to be a principal regulator of in vivo immune responses. Despite extensive biochemical characterization of the p50-55 (a) and p70-75 (/3) chains of the IL-2 receptor, molecular cloning and sequencing of their genes, and the reconstitution of high-affinity receptors in heterologous systems through gene transfer (1-5), few details are available regarding the mechanism by which the IL-2 signal is transmitted from the surface of the cell to its interior. Unlike many other growth factor receptors that possess ligand-dependent, tyrosine-specific kinase activity, neither the a chain nor the 3 chain of the IL-2 receptor appears to have intrinsic kinase activity (4, 6, 7). Also, none of the known second messenger pathways that regulate kinase activity through the production of cyclic nucleotides or elevations in cytosolic Ca2' appear to be directly involved in IL-2 signal transduction (for reviews, see refs. 8 and 9). And yet, both tyrosine and serine/threonine phosphorylation of intracellular proteins are rapid events in IL-2-stimulated T cells (10)(11)(12)(13)(14). Clearly, therefore, IL-2 and its receptor must regulate the activity of kinases in lymphocytes, but it has remained obscure as to which ones are involved.Here, we demonstrate that IL-2 specifically induces the phosphorylation and activation, in an IL-2-dependent T-cell clone, CTLL-2, of the p72-74 kinase encoded by the c-Raf-1 protooncogene. This serine/threonine-specific kinase has recently been implicated in signal-transduction events mediated by several tyrosine kinase growth factor receptors and oncogene products (15, 16). Gene transfer experiments using v-raf have previously suggested...

Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1

“…particularly in cells of hematopoietic origin (Carroll et al, 1991;Turner et al, 1991), in cells transformed by oncogenic Ras (Jelinek et al, 1996) and in cells exposed to ionizing radiation (Kasid et al, 1996). Furthermore, experiments in both mammalian and Sf9 cells have implicated two adjacent tyrosine residues in the catalytic domain of Raf-1 (Y340 and Y341) as being important for the regulation of Raf-1 kinase activity.…”

The role of Stat5a and Stat5b in signaling by IL-2 family cytokines

“…As is typical of type I cytokine receptors, neither IL-2Rb nor g c contains protein kinase activity in its cytoplasmic domain (Hatakeyama et al, 1989;Takeshita et al, 1992). However, within minutes following the interaction of IL-2 with the high-or intermediate-a nity IL-2 receptors, a number of cellular proteins, including IL2Rb and g c , are phosphorylated on tyrosine residues (Farrar and Ferris, 1989;Turner et al, 1991), at least in part by the receptor-associated protein tyrosine kinases, Jak1 and Jak3 (Friedmann et al, 1996;Johnston et al, 1994;Witthuhn et al, 1994). It has been demonstrated that in the absence of IL-2, Jak1 is associated with IL-2Rb Russell et al, 1994;Witthuhn et al, 1994) and Jak3 is with g c Russell et al, 1994;Figure 2).…”

“…An important pathway activated following stimulation by IL-2 is the Ras-Raf-MAP kinase pathway (Graves et al, 1992;Turner et al, 1991Turner et al, , 1993Zmuidzinas et al, 1991; Figure 2). The adaptor protein Shc associates with Tyr-338 of IL-2Rb via its PTB domain and it is tyrosine phosphorylated by Jak1 (Friedmann et al, 1996;Ravichandran et al, 1996).…”

Cloning of Human Stat5B

“…In addition to activating a variety of tyrosine kinases (2), IL-2 also activates other signaling molecules, including serine kinases (50) and Ras (51). The COS-7 reconstitution system reported herein may be valuable in screening transfected gene products to help identify other signaling molecules required for mediating IL-2-dependent, Stat5-mediated transcription and in clarifying whether Stat5A homodimers, Stat5B homodimers, and Stat5A-Stat5B heterodimers regulate the same or different genes.…”