Abstract
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a central role in the intracellular signaling of P2X7 nucleotide receptors in neurons and glial cells. Fine spatio-temporal tuning of mitogen-activated protein (MAP) kinases is essential to regulate their biological activity. MAP kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues in MAP kinases. This study focuses on how DUSP, DUSP6/MKP3, a phosphatase specific for ERK1/2 is regulated by the P2X7 nucleotide receptor in cerebellar granule neurons and astrocytes. Stimulation with the specific P2X7 agonist, BzATP, or epidermal growth factor (EGF) (positive control for ERK activation) regulates the levels of DUSP6 in a time dependent manner. Both agonists promote a decline in DUSP6 protein, reaching minimal levels after 30 min yet recovering to basal levels after 1 h. The initial loss of protein occurs through proteasomal degradation, as confirmed in experiments with the proteasome inhibitor, MG-132. Studies carried out with Actinomycin D demonstrated that the enhanced transcription of the Dusp6 gene is responsible for recovering the DUSP6 protein levels. Interestingly, ERK1/2 proteins are involved in the biphasic regulation of the protein phosphatase, being required for both the degradation and the recovery phase. We show that direct Ser197 phosphorylation of DUSP6 by ERK1/2 proteins could be part of the mechanism regulating their cytosolic levels, at least in glial cells. Thus, the ERK1/2 activated by P2X7 receptors exerts positive feedback on these kinase’s own activity, promoting the degradation of one of their major inactivators in the cytosolic compartment, DUSP6, both in granule neurons and astrocytes. This feedback loop seems to function as a common universal mechanism to regulate ERK signaling in neural and non-neural cells.
Highlights
The mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) regulate multiple processes in neural cells, such as the balance between survival and cell death, proliferation and differentiation, and they play a crucial role during nervous system development and in neuroregeneration
We previously demonstrated that stimulation of the P2X7 receptor in cerebellar granule neurons and astrocytes was associated with Extracellular signal regulated kinases (ERK) activation quantified in western blots probed with phospho-specific antibodies (Jiménez et al, 2002; Ortega et al, 2011)
In the present study we demonstrate the presence of the dual specificity protein phosphatase DUSP6 in granule neurons and astrocytes from the rat cerebellum, and its regulation by nucleotide P2X7 and epidermal growth factor (EGF)
Summary
The mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) regulate multiple processes in neural cells, such as the balance between survival and cell death, proliferation and differentiation, and they play a crucial role during nervous system development and in neuroregeneration. Numerous factors modulate ERK1/2 signaling, alone or in combination, including extracellular signals, scaffolding and signaling proteins recruited to specific cascades, and other intrinsic tissue or cell factors. The spatio-temporal regulation of the ERK1/2 pathway shapes the final biological response to its activation. Transient or sustained early activation of ERK1/2 signaling is involved in either proliferation or differentiation, respectively. By contrast, prolonged and delayed ERK1/2 activation is more closely related to cell death following certain toxic stimuli (Marshall, 1995; Chu et al, 2004). Fine tuning of ERK1/2 signaling is necessary to obtain adequate responses
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