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Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular
V Cottin1, A Van Linden, D W Riches
1Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Abstract:
The interaction of tumor necrosis factor-alpha (TNFalpha) with its receptor sets in motion downstream signaling events including the activation of members of the mitogen-activated protein kinase (MAPK) family. In this study, we show that p42(mapk/erk2) phosphorylates sequences present within the cytoplasmic domain of CD120a (p55). By using a GST-CD120a-(207-425) fusion protein as substrate, phosphorylation was induced following stimulation of mouse macrophages with TNFalpha, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and zymosan particles and was blocked by immunodepletion of p42(mapk/erk2) and by specific inhibition of p42(mapk/erk2) activation with PD098059. Transfection of COS-7 cells with CD120a (p55), wild-type p42(mapk/erk2), and constitutively active MEK-1 followed by metabolic labeling with [(32)P]orthophosphate indicated that p42(mapk/erk2) phosphorylated the cytoplasmic domain of CD120a (p55) in intact cells. As a consequence of phosphorylation, CD120a (p55) expression at the plasma membrane and Golgi apparatus was lost and the receptor accumulated in intracellular tubular structures associated with the endoplasmic reticulum. Mutation of the four Ser and Thr ERK consensus phosphorylation sites to Ala residues inhibited the ability of the receptor to redistribute to intracellular tubules in a p42(mapk/erk2)-dependent fashion; whereas mutation of the phosphorylation sites to Asp and Glu residues mimicked the effect of receptor phosphorylation. These findings thus indicate that the phosphorylation of CD120a (p55) alters the subcellular localization of the receptor and may thereby result in changes in its signaling properties.
Insights
Tumor necrosis factor-alpha (TNFalpha) signaling activates mitogen-activated protein kinase (MAPK) family members. This study shows p42(MAPK/ERK2) phosphorylates CD120a (p55), altering its subcellular localization and potentially its signaling properties.
Area of Science:
- Cellular signaling pathways
- Receptor biology
- Protein phosphorylation
Background:
- Tumor necrosis factor-alpha (TNFalpha) interaction with its receptor initiates downstream signaling, including mitogen-activated protein kinase (MAPK) activation.
- The precise mechanisms by which TNFalpha receptor signaling influences cellular localization and function require further elucidation.
Purpose of the Study:
- To investigate the role of p42(MAPK/ERK2) in the phosphorylation of the TNFalpha receptor CD120a (p55).
- To determine the functional consequences of CD120a (p55) phosphorylation on its subcellular localization and potential signaling.
Main Methods:
- Utilized a GST-CD120a-(207-425) fusion protein as a substrate to assess phosphorylation in response to various stimuli.
- Employed immunodepletion and specific inhibitors (PD098059) to confirm the involvement of p42(MAPK/ERK2).
- Investigated phosphorylation in intact cells using COS-7 transfection, MEK-1 activation, and metabolic labeling with [(32)P]orthophosphate.
- Analyzed the impact of site-directed mutagenesis of phosphorylation sites on receptor localization.
Main Results:
- p42(MAPK/ERK2) was confirmed to phosphorylate the cytoplasmic domain of CD120a (p55) in response to TNFalpha and other stimuli.
- Phosphorylation led to the loss of CD120a (p55) from the plasma membrane and Golgi apparatus, with accumulation in intracellular tubular structures.
- Mutating phosphorylation sites to alanine blocked this redistribution, while mutation to aspartate/glutamate mimicked the effect, confirming phosphorylation's role.
Conclusions:
- Phosphorylation of CD120a (p55) by p42(MAPK/ERK2) is a key event following TNFalpha receptor activation.
- This phosphorylation event significantly alters the subcellular localization of the CD120a (p55) receptor.
- Changes in receptor localization due to phosphorylation may lead to modulation of downstream signaling properties.