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Biochemical analysis of MEK activation in NIH3T3 fibroblasts. Identification of B-Raf and other activators
C W Reuter1, A D Catling, T Jelinek
1Department of Microbiology, University of Virginia Health Sciences Center, School of Medicine, Charlottesville 22908, USA.
Abstract:
Numerous potential activators of MEK have been identified, including c-Raf-1, B-Raf, c-Mos, and a family of MEK kinases. However, little information gives insight into the activators actually utilized in vivo. To address this, we have used column chromatography and a coupled MEK activation assay to identify in NIH3T3 cells, two major MEK activators, and a third insulin-specific activator. The first MEK activator has an apparent M(r) of 40,000-50,000, was immunologically distinct from A-Raf, B-Raf, c-Raf-1, c-MEKK, c-Mos, MEK1, and MEK2, and was rapidly activated by serum, platelet-derived growth factor (PDGF), insulin, thrombin, and phorbol ester. The second MEK activator was identified as B-Raf. Activation of 93-95 kDa B-Raf was observed in column fractions and B-Raf immunoprecipitates from cytosolic and particulate fractions after stimulation with serum or PDGF, but not insulin. c-Raf-1 from cytosol did not exhibit MEK activator activity; however, c-Raf-1 immunoprecipitates from the particulate fraction revealed MEK activator activity that was enhanced after stimulation with PDGF or phorbol ester, but not serum or insulin. Both c-Mos and c-MEKK were present in NIH3T3 fibroblasts but did not show MEK activator activity. These data provide direct evidence that 93-95-kDa B-Raf isozymes and an unidentified 40-50-kDa MEK activator are major agonist-specific MEK activators in NIH3T3 fibroblasts.
Insights
Researchers identified two major MEK activators in NIH3T3 cells: B-Raf and an unknown 40-50 kDa protein. These findings clarify in vivo MEK activation pathways, crucial for cell signaling research.
Area of Science:
- Cellular signaling pathways
- Protein kinase regulation
- Molecular biology
Background:
- Mitogen-activated protein kinase kinase (MEK) is a key regulator of cellular processes.
- Potential activators like c-Raf-1, B-Raf, and c-Mos have been identified, but in vivo relevance is unclear.
Purpose of the Study:
- To identify and characterize the in vivo activators of MEK in NIH3T3 fibroblasts.
- To differentiate between known and novel MEK activators utilized by cells.
Main Methods:
- Column chromatography was employed to isolate cellular components.
- A coupled MEK activation assay was used to detect MEK activator activity.
- Immunological assays confirmed the identity of specific proteins.
Main Results:
- Two major MEK activators were identified: B-Raf (93-95 kDa) and an unknown protein (40-50 kDa).
- The 40-50 kDa activator was distinct from known Raf and MEK kinases and activated by various growth factors and stimuli.
- B-Raf activation was observed with serum and PDGF, while c-Raf-1 showed activity in particulate fractions upon PDGF stimulation.
Conclusions:
- B-Raf and the novel 40-50 kDa protein are major MEK activators in NIH3T3 cells.
- These activators exhibit agonist-specific activation patterns, highlighting distinct roles in cellular signaling.