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Ras-dependent growth factor regulation of MEK kinase in PC12 cells
C A Lange-Carter1, G L Johnson
1Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
Abstract:
Mitogen-activated protein kinases (MAPKs) are rapidly activated in response to stimulation of diverse receptor types. MAPKs are positively regulated by phosphorylation on threonine and tyrosine by MAP kinase or extracellular signal-regulated kinase (ERK) kinases (MEKs). MEK kinase (MEKK) is part of a family of serine-threonine protein kinases that phosphorylate and activate MEKs independently of Raf. MEKK was rapidly and persistently activated in response to stimulation of resting PC12 cells with epidermal growth factor (EGF). Nerve growth factor (NGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated MEKK, although to a lesser degree than did EGF. Activation of MEKK and B-Raf in response to EGF was inhibited by expression of dominant negative N17Ras. Expression of oncogenic Ras resulted in activation of MEKK. Stimulation of synthesis of cyclic adenosine 3',5'-monophosphate abolished activation of MEKK and B-Raf by EGF, NGF, and TPA. Thus, Ras simultaneously controls the activation of members of the Raf and MEKK families of protein kinases.
Insights
Ras protein simultaneously controls the activation of Raf and MEK kinase (MEKK) families. This signaling pathway is crucial for cellular responses to growth factors like epidermal growth factor (EGF).
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Signal transduction
Background:
- Mitogen-activated protein kinases (MAPKs) are key regulators of cellular processes.
- MAPKs are activated by phosphorylation by upstream kinases, including MEK kinases (MEKKs).
- MEKKs are serine-threonine protein kinases that activate MEKs, independent of Raf kinases.
Purpose of the Study:
- To investigate the role of Ras in the activation of MEKK and Raf kinase families.
- To understand the signaling mechanisms downstream of growth factor receptors.
Main Methods:
- Stimulation of PC12 cells with epidermal growth factor (EGF), nerve growth factor (NGF), and 12-O-tetradecanoylphorbol-13-acetate (TPA).
- Expression of dominant-negative N17Ras and oncogenic Ras.
- Measurement of MEKK and B-Raf activation.
- Modulation of cyclic adenosine 3',5'-monophosphate levels.
Main Results:
- MEKK was rapidly and persistently activated by EGF, and to a lesser extent by NGF and TPA.
- EGF-induced activation of MEKK and B-Raf was inhibited by dominant-negative N17Ras.
- Oncogenic Ras expression led to MEKK activation.
- Elevated cyclic adenosine 3',5'-monophosphate levels abolished MEKK and B-Raf activation by all tested stimuli.
Conclusions:
- Ras signaling simultaneously regulates both Raf and MEKK kinase families.
- Ras acts as a central mediator in growth factor-induced signaling pathways.
- This study elucidates a key convergence point in cellular signal transduction.