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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 N-terminal 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.

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulates the activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase (MAPK), and S6 protein kinase (S6K) activities both in a time- and dose-dependent manner. A single peak of MAPKK activity, four peaks of activity against the S6 synthetic peptide, RRLSSLRA (S6 peptide), and three distinct peaks toward myelin basic protein (MBP) were observed after Mono-Q chromatography of LIF-stimulated cell extracts. Two of the MBP kinase activities correlated with the stimulation of extracellular signal-regulated kinases 1 and 2. Interestingly, down-regulation of protein kinase C (PKC) by chronic treatment of 3T3-L1 cells with phorbol ester was found to attenuate, but not block, the LIF-mediated stimulation of MAPKK, MAPK, and S6K activities in 3T3-L1 cells. Treatment of 3T3-L1 cells with epidermal growth factor increased MAPKK, MAPK, and S6K activities to a similar extent as LIF, but this activation was not attenuated by down-regulation of PKC. Our results suggest that the full activation of the MAPK cascade by LIF may require inputs from multiple signaling pathways, one of which is dependent upon the presence of functional PKC.