Phosphorylation of the Membrane Proximal Region of Tumor Necrosis Factor Receptor CD120a (p55) at ERK Consensus Sites

Abstract

The interaction of tumor necrosis factor-␣ with its receptor CD120a (p55) initiates downstream signaling cascades that include the activation of the mitogen-activated protein kinase (MAPK), p42 mapk/erk2 . The membrane proximal region of CD120a (p55) is Ser-, Thr-, and Pro-rich and contains four mitogen-activated protein kinase consensus phosphorylation sites. In recent work, we showed that CD120a (p55) itself is a target of phosphorylation by p42 mapk/erk2, and after phosphorylation, the receptor is redistributed from the cell surface and Golgi complex to intracellular tubular structures associated with elements of the endoplasmic reticulum. The goal of this study was to define the specific amino acid residues that are phosphorylated. Deletional mutagenesis of the cytoplasmic domain of CD120a (p55) indicated that two sites located between residues 207-254 and 250 -300 were phosphorylated predominantly on Thr and Ser residues, respectively. Site-directed mutagenesis of Ser and Thr residues contained within the extracellular signal-regulated kinase (ERK) consensus sequences indicated that the preferred residues were Thr-236 and Ser-270. Primary phosphorylation at these sites appeared to enable subsequent phosphorylation at Ser-240 and Ser-244, although the level of phosphorylation of these latter two sites was less than the preferred sites. Through the use of specific ligation of CD120a (p55) alone and mice deficient in CD120a (p55), CD120b (p75), or both receptors, CD120a (p55) was shown to be necessary and sufficient for the induction of kinase activity. These findings thus suggest that the phosphorylation of Thr-236 and Ser-270 within the membrane proximal region of CD120a (p55) are the preferred sites of phosphorylation by p42 mapk/erk2 and may set in motion phosphorylation at other sites.

A Structure-Function Analysis of Serine/Threonine Phosphorylation of the Thrombopoietin Receptor, c-Mpl

“…For example, T69/S61A/S62A and T69/S64A/ T65A cells lost the same radiolabeled phosphopeptides in mapping experiments, suggesting that phosphorylation of residues Ser-61, Ser-62, and/or Ser-64 is dependent on one or two of the other Ser(P) residues. This result is similar to that with Ser/ Thr phosphorylation of the p55 TNF receptor, where phosphorylation of Thr-236 and Ser-270 enable subsequent phosphorylation of Ser-240 and Ser-244 (15). Similar hierarchical and synergistic phosphorylation events were also reported for rhodopsin and heat shock factor-1 (16,17).…”

“…4F). It is possible that like the p55 tumor necrosis factor (TNF) receptor, heat shock factor-1, and rhodopsin proteins (15)(16)(17), phosphorylation of one Ser residue might be responsible for phosphorylation of subsequent Ser residues, helping to explain why the same spots were lost in different mutations. Nevertheless, the results in Fig.…”

Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling in Activated T Cells Abrogates TRAIL-Induced Apoptosis Upstream of the Mitochondrial Amplification Loop and Caspase-8

“…A protein synthesis-independent signal-based mechanism that regulates TNF-R1 sequestration has been reported. In this case, MAPK/ERK signaling was shown to phosphorylate TNF-R1 directly, thereby causing the internalization of the receptor from the surface of the cell to the cytosol and inhibition of its cytotoxic ability (51,52). However, our study excludes the possibility of TRAIL-R internalization or down-regulation as a MAPK/ERK target, because the surface expression of the DR4 and DR5 was not altered by the activation of MAPK/ERK.…”

Oxysterols Inhibit Phosphatidylcholine Synthesis via ERK Docking and Phosphorylation of CTP:Phosphocholine Cytidylyltransferase

“…Furthermore, this pattern is highly consistent with the kinetics of ERKs, as they regulate a variety of physiological readouts such as platelet-derived growth factor-induced mitogenesis (31), cellular propagation of influenza (32), leukotactin-1 control of cell cycle progression (33), and effects of phorbol esters and nerve growth factors on cell survival (34,42). Although the data favor 22-HC and 9-cis-RA activation of p42/44 kinase, concurrent activation of p38 kinase, JNK, or p34 cdc2 is also possible, as these enzymes utilize similar minimal recognition motifs ((Ser/Thr)-Pro) for substrate phosphorylation (43,44). On the other hand, because (Ser/Thr)-Pro sequences are fairly ubiquitous within substrates, the specificity and prevention of inappropriate cross-talk between related kinases are provided by assembly of scaffolding proteins and interaction with docking regions on target proteins (45).…”