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Tyrosine phosphorylation of extracellular signal-regulated protein kinase 4 in response to growth factors
X Peng1, J M Angelastro, L A Greene
1Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Abstract:
Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this re-agent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.
Insights
Researchers identified ERK4, a new member of the ERK protein family, in rat cells. This protein is activated by growth factors like NGF and EGF, suggesting a unique role in cell signaling pathways.
Area of Science:
- Cellular Biology
- Molecular Biology
- Signal Transduction
Background:
- Extracellular signal-regulated protein kinases (ERKs) are crucial in mitogen-activated protein kinase signaling.
- ERK1 and ERK2 are the most studied ERK forms, while other family members remain less understood.
- ERK4 was previously identified based on antibody cross-reactivity but lacked detailed characterization.
Purpose of the Study:
- To characterize a previously identified ERK4 protein in rat PC12 cells.
- To investigate the activation pathways and kinetics of ERK4 in response to various stimuli.
- To determine the distinct identity and potential role of ERK4 within the ERK family.
Main Methods:
- Immunoprecipitation of ERK4 using anti-ERK antiserum R2 in rat PC12 cells.
- Analysis of tyrosine phosphorylation in response to nerve growth factor (NGF), epidermal growth factor (EGF), insulin, and cyclic AMP.
- Investigation of the ras-dependent pathway for ERK4 activation.
- Two-dimensional gel electrophoresis to distinguish ERK4 from ERK1 isoforms.
Main Results:
- ERK4 was successfully immunoprecipitated and characterized in rat PC12 cells.
- ERK4 undergoes rapid tyrosine phosphorylation upon stimulation with NGF and EGF, and to a lesser extent with insulin and cyclic AMP.
- ERK4 activation by NGF and EGF is ras-dependent, with distinct kinetics for NGF compared to EGF.
- ERK4 is confirmed as a distinct member of the ERK family, separate from ERK1 isoforms.
Conclusions:
- ERK4 represents a novel, growth factor-responsive member of the ERK protein kinase family.
- ERK4 activation pathways share similarities with ERK1/ERK2, including ras-dependency.
- The distinct characteristics of ERK4 suggest it may play a unique role in intracellular signaling cascades.