Phosphorylation of Human gp130 at Ser-782 Adjacent to the Di-leucine Internalization Motif

Abstract

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF-and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately Nterminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide. Leukemia inhibitory factor (LIF)1 is a multifunctional cytokine that mediates a variety of physiological effects in numerous cell lineages. For example, LIF stimulates leukemic cell differentiation and the proliferation of myeloid and platelet precursors, inhibits the differentiation of embryonic stem cells, stimulates acute phase protein synthesis in hepatocytes, inhibits lipoprotein lipase activity in adipocytes, stimulates myoblast proliferation, and converts sympathetic neurons from noradrenergic to cholinergic phenotypes (Refs. 1-3 and the references therein). LIF, ciliary neurotrophic factor, oncostatin M, interleukin-11, cardiotrophin-1, and interleukin-6 make up a distinct subgroup of the cytokine superfamily that all exhibit a four-anti-parallel ␣-helical bundle structure arranged in a two-up, two-down configuration and associate with the shared signaling molecule, gp130, after initial binding to their unique low affinity ␣-receptor binding subunits (1-4).LIF signals through a heterodimeric receptor complex consisting of LIFR and gp130 (5). Lacking intrinsic enzymatic activity (6), initiation of LIF receptor signaling occurs in a manner analogous to that of activated growth factor receptors (7, 8). LIF-stimulated dimerization of LIFR and gp130 results in activation of the Jak/Tyk family of nonreceptor protein tyrosine kinases (4, 9). The activated Jaks then phosphorylate and activate the signal transducers and activators of transcription (STATs), which regulate a variety of cytokine-responsive genes (10 -13). Activated LIF receptors have also been shown to recruit and stimulate numerous SH2-containing signaling molecules, including phospholipase C-␥, Shc, Grb2, phosphoinositol 3-kinase, pp120, and the protein-tyrosine phosphatase SHP-2 (14).Activated LIF receptors also stimulate several components of the MAP kinase cascade, including MAPK kinase, the MAPK isozymes ERK1 and ERK2, and ...

Discovery of a Novel Orally Active Small-Molecule gp130 Inhibitor for the Treatment of Ovarian Cancer

“…S7). The role of phospho-S782 in gp130 was first documented in 2000 to downregulate gp130 cell surface expression (5). Recently, it has been reported that phospho-S782-associated downregulation of gp130 cell surface expression is mediated by immediate internalization (46).…”

“…Ligand binding induces the association of gp130 with a cytokine-specific receptor-a chain, followed by the activation of downstream signaling cascades including JAK/STAT, RAS/ RAF/MAPK, and PI3K/AKT pathways (4). It has been shown that phosphorylation of gp130 at Ser782 downregulates cell surface expression of gp130 (5,6). As a ubiquitously expressed receptor, gp130 is involved in a wide range of important biologic processes including inflammation, autoimmunity, cancer (7), stem cell maintenance, and embryonic development (8).…”

Tyrosine phosphorylation of scavenger receptor cysteine‐rich WC1 is required for the WC1‐mediated potentiation of TCR‐induced T‐cell proliferation

“…A similar motif is contained in the cytoplasmic tails of CD3g, gp130, and CD4. Phosphorylation of an upstream serine and the interaction of the two leucines with clathrin-associated adapter protein AP-2 complexes are required for endocytosis [37][38][39]. As in WC1, the internalization motif in the CD4 cytoplasmic tail is located in a predicted alpha-helix.…”

Oncostatin M Regulates the Synthesis and Turnover of gp130, Leukemia Inhibitory Factor Receptor α, and Oncostatin M Receptor β by Distinct Mechanisms

“…The other receptor subunits, gp130 and OSMR␤, do not possess a phosphorylation site for ERK 1/2 and, thus, do not appear to be appreciably influenced by activated ERK. However, serine 782 of gp130 located in the cytoplasmic domain has been described as being phosphorylated and directing the cell surface expression of the receptor subunit (14). The kinase for this modification is still unknown.…”