Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines.

Abstract

By immunoblotting with antibodies for phosphotyrosine, we have demonstrated that the hematopoietic growth factors interleukin-2, interleukin-3, interleukin-4, and granulocyte-macrophage colony-stimulating factor stimulate the tyrosine phosphorylation of specific sets of proteins in murine hematopoietic progenitor cell lines. The stimulation of tyrosine phosphorylation is a receptor-dependent transient event. The effect of these hematopoietic growth factors on protein tyrosine phosphorylation was not mediated through protein kinase C.The growth and differentiation of hematopoietic cells is controlled by a number of glycoproteins known as interleukins or colony-stimulating factors (CSFs) (for reviews, see references 4 and 23). The actions of these factors are mediated through specific receptors present on the surface of hematopoietic cells of specific lineages and stages of differentiation (4, 23). Many of the genes encoding these growth factors and a few of their receptors have been cloned; however, the intracellular mechanisms of action of these factors are largely unknown. Activation of protein tyrosine phosphorylation is an integral response in many different mitogenic systems (2). The receptors for several peptide growth factors, e.g., the epidermal growth factor and CSF-1, contain tyrosine kinase domains which reside in the intracellular portions of the receptor proteins (2). The receptor tyrosine kinase activity is essential for the transduction of mitogenic signals (3,14). In contrast to the tyrosine kinase receptors, the two interleukin-2 (IL-2) receptors are smaller (55 and 70 kilodaltons [kDa]), and the 55-kDa receptor has been shown to have only a 13-amino-acid intracellular domain (6,8,19,30,(35)(36)(37). By chemical cross-linking with radioactive ligands, putative receptors for IL-3 and for granulocyte-macrophage CSF (GM-CSF) have been found to have apparent subunit molecular weights of 60 to 75 and 51 kDa, respectively (29,31,38); these receptors may also contain small intracellular domains. Although the receptors for growth factors such as IL-2, IL-3, and GM-CSF may not contain tyrosine kinase domains, it does not rule out the possibility that these growth factors also activate protein tyrosine phosphorylation in their signal transduction pathways.To examine the effects of IL-2, IL-3, and GM-CSF on protein tyrosine phosphorylation, we used two murine hematopoietic cell lines, IC-2.9 and 32Dcl5, which were originally established as IL-3-dependent cell lines. IC-2.9 is a clone of the mast cell line IC-2 (16), which can proliferate on IL-3, IL-4, or GM-CSF (17). 32Dcl5 (32D [12]) is a basophilic cell line which expresses low levels of IL-2 receptors. Le Gros et al. found that the number of IL-2 receptors in 32D cells can be increased by culturing the cells in IL-2 alone and that the resulting 32D/IL-2 cells can proliferate on either IL-3 or IL-2 (18). Exponentially growing IC-2.9 or 32D/IL-2 * Corresponding author. cells were washed free of serum and growth factors and incubated in starvation medium (...

Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.

“…A set of proteins is phosphorylated rapidly on tyrosine residues after the addition ofthese growth factors (21)(22)(23)(24)(25) (Fig. 6).…”

CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand).

“…The PI-PLC-activated CD3/Ti stimulation requires tyrosine phosphorylation (37), and CD45 ligation prevents tyrosine phosphorylation of specific substrates during either CD2-or CD3-mediated activation (17). Stimulation of some of the receptors studied in the present report, including those for IL3, GM-CSF, and MGF, induces rapid tyrosine phosphorylation of cellular substrates (38). Phospholipase C y can be activated as one of these substrates by epidermal growth factor and platelet-derived growth factor receptors (39,40) .…”

Enhancement of ovine trophoblast interferon by granulocyte macrophage-colony stimulating factor: possible involvement of protein kinase C

“…They exert their effects on target cells through specific receptors, and c subunits, that are regarded as members of the Jak family (Ihle & Kerr 1995). Although the GM-CSF and IL-3 receptors do not contain intrinsic tyrosine kinase activity, many cellular proteins are rapidly tyrosine phosphorylated in response to GM-CSF and/or IL-3 (Morla et al 1988, Sorensen et al 1989, Isfort & Ihle 1990, Duronio et al 1992. It has been shown that the human c subunit mediates activation of Jak2 kinase (Darnell et al 1994, Quelle et al 1994, Rothman et al 1994 and Ras, Raf-1, and mitogen activated protein kinase (MAPK) (Sato et al 1993.…”