Phosphorylation of the membrane proximal region of tumor necrosis factor receptor CD120a (p55) at ERK consensus sites

The interaction of tumor necrosis factor-alpha with its receptor CD120a (p5 5) initiates downstream signaling cascades that include the activation of t he mitogen-activated protein kinase (MAPH), p42(mapk/erk2), The membrane pr oximal region of CD120a (p55) is Ser-, Thr-, and Pro-rich and contains four mitogen-activated protein kinase consensus phosphorylation sites. In recen t work, we showed that CD120a (p55) itself is a target of phosphorylation b y 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 st udy 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 phosphory lated predominantly on Thr and Ser residues, respectively. Site-directed mu tagenesis of Ser and Thr residues contained within the extracellular signal -regulated kinase (ERK) consensus sequences indicated that the preferred re sidues were Thr-236 and Ser-270, Primary phosphorylation at these sites app eared to enable subsequent phosphorylation at Ser-240 and Ser-244, although the level of phosphorylation of these latter two sites was less than the p referred 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 pre ferred sites of phosphorylation by p4(2mapk/erk2) and may set in motion pho sphorylation at other sites.