Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

Abstract

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose-and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor a-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokinestimulated expression of a reporter gene under the control of the ,8-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism ofheterologous receptor regulation ofLIFR signaling occurring at Ser-1044.Leukemia inhibitory factor (LIF) is a member of the family of multifunctional cytokines capable of stimulating numerous physiological processes in a variety of cells (1, 2). LIF, along with ciliary neurotrophic factor, interleukins (ILs) 6 and 11, and oncostatin M, constitutes a distinct subgroup of the cytokine family (3) in which each member has its own unique a-receptor subunit that associates with the shared receptor subunit gp130 to initiate transmembrane signaling (4-12).Early studies of LIF signaling have indicated activation of a pathway(s) involving both Tyr and Ser/Thr protein kinases (12, 13). Both the low-affinity LIF receptor a subunit (LIFR) and gpl3O associate with and stimulate members of the Jak/Tyk family of nonreceptor protein-tyrosine kinases (14). We (15) and others (16,17) have shown recently that activation of LIFR by agonist rapidly stimulates several components of the mitogen-activated protein kinase (MAPK) cascade, including MAPK kinase, the MAPK isozymes extracellular signal-regulated kinases (ERKs) 1 and 2, and S6 protein kinase activities against both S6 peptide and 40S ribosomes. In the current study, we show that the human LIFR can be phosphorylated at Ser-1044 by activated MAPK in vitro and suggest that this residue mediates a pathway for regulation of LIFR signaling after stimulation of a heterologous receptor system.

Inhibition of IL-6 and IL-10 Signaling and Stat Activation by Inflammatory and Stress Pathways

“…In contrast, comparable inhibition by CA-MKK3, CA-MKK6, and CA-MEK1 was seen when LIF, which signals through gp130 and a LIF-specific ␣-chain, was used. This is consistent with previously described inhibition of LIF signaling by ERKs (57) and confirms that CA-MEK1 is indeed active in our system. The inhibitory effect of CA-MEK1 or CA-MKK3 and CA-MKK6 was not detected when IFN-␥ was used, suggesting that these kinases do not nonspecifically block all Jak-Stat signaling, consistent with the lack of inhibition of IFN-␥ Stat1 activation (Fig.…”

Stimulation of Leukemia Inhibitory Factor Receptor Degradation by Extracellular Signal-regulated Kinase

“…Plasmid Constructs-The expression vectors for the wild type and S1044A (S185A) mutant human LIFR␣ and chimeric G-CSFR-LIFR␣ in pDC302 have been described (10,22). Cytoplasmic domains of these receptors were modified by the addition of Myc epitope (EQKLI-SEEDLN) to the C terminus.…”

“…Moreover, activation of ERK by insulin also increases LIFR␣ Ser 185 phosphorylation. It has been suggested that this modification may contribute to the modulation by insulin of cell responsiveness to LIF (22). However, the mechanism of this LIFR␣ regulation is unknown.…”

“…ERK1 and ERK2 are implicated in phosphorylating Ser 1044 (or Ser 185 of the cytoplasmic domain) of LIFR␣ (22). Moreover, activation of ERK by insulin also increases LIFR␣ Ser 185 phosphorylation.…”

Functional Map of a Placenta-specific Enhancer of the Human Leukemia Inhibitory Factor Receptor Gene

“…The LIFR was found to contain 1097 amino acid residues (a 44-residue signal sequence, a 789-residue extracellular domain, a 26-residue transmembrane domain, and a 238-residue cytoplasmic domain) (6), and exhibits the characteristic structure of this receptor family, including two folding domains in the extracellular region and a Trp-Ser-X-Trp-Ser motif in the Cterminal (1). The LIFR heterodimerizes with gp130 to mediate intracellular signaling of interleukin-6 (IL-6) cytokine family members and is a substrate for mitogen-activated protein kinase (7)(8)(9)(10)(11)(12). The phenotype of the LIFR knockout mouse demonstrates that LIFR is essential for animal survival (13,14), whereby homozygote animals died within 24 h of birth (13,14), and severely affected tissues include placenta, bone, liver, and neurons (13).…”