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[Constitutive activity of MAP kinase cascades in REF cells transformed by E1A and cHa-ras oncogenes]
S B Svetlikova1, M V Abramova, A N Kukushkin
1Institute of Cytology, RAS, St. Petersburg.
Abstract:
Proteins of Ras family play an important role in regulation of cell growth and proliferation, and their mutations can lead to growth factor-independent proliferation due to constitutive activity of various signal transduction cascades. In the present work, we studied the activity of ERK, JNK and p38 MAP-kinase cascades in rat embryo fibroblast cells transformed with oncogenes E1A and cHa-ras. These transformed cells are characterized by a high and non-regulated activity of transcription factor AP-1 involved in the regulation of cell proliferation. Since phosphorylation of AP-1 depends on the activity of relevant MAP-kinase cascades (ERK, JNK and p38), we analysed the expression of non-phosphorylated forms of the kinases and their phosphorylated state in E1A + cHa-ras cells using antibodies specific to non-phosphorylated and phosphorylated proteins. It has been established that transformed cells contain higher amounts of non-phosphorylated ERK, JNK and p38 kinases, thus implying a reduced degradation of these and other proteins in the transformants. The content of phosphorylated (active) forms studied in Western blot-analysis with phosphoantibodies was shown to be also higher in exponentially growing E1A + cHa-ras cells. But serum stimulation of the starved cells gave insignificant rise to an increase of ERK, JNK and p38 phosphorylation. Nevertheless, an in vitro kinase assay performed with the kinases, either immunoprecipitated by antibody or bound to GST-fusion substrates, enabled us to show a certain level of stimulation of c-Jun-associated (JNK) and MEF2A-associated (p38) kinase activity in serum stimulated E1A + cHa-ras cells. Thus, the obtained results show that transformation of fibroblasts with E1A and ras oncogenes may contribute to constitutive activation of ERK, JNK and p38 kinase cascades responsible for a high and non-regulated activity of MAP-kinase-dependent transcription factors, in particular AP-1.
Insights
Ras oncogenes like E1A and cHa-ras drive uncontrolled cell growth by constitutively activating ERK, JNK, and p38 MAP-kinase cascades. These kinases lead to high AP-1 activity, promoting cell proliferation.
Area of Science:
- Cellular signaling pathways
- Oncogene-induced transformation
- Signal transduction cascades
Context:
- Ras family proteins regulate cell growth and proliferation.
- Mutations in Ras proteins can lead to uncontrolled cell proliferation.
- Aberrant signaling pathways are implicated in cancer development.
Purpose:
- To investigate the activity of ERK, JNK, and p38 MAP-kinase cascades in rat embryo fibroblast cells transformed by E1A and cHa-ras oncogenes.
- To analyze the expression and phosphorylation status of these kinases in transformed cells.
- To understand the role of these kinases in the constitutive activation of transcription factor AP-1.
Summary:
- Transformed cells exhibited higher levels of both non-phosphorylated and phosphorylated ERK, JNK, and p38 kinases.
- Serum stimulation showed limited impact on overall kinase phosphorylation but induced activity in JNK and p38 in vitro.
- Results suggest E1A and ras oncogene transformation leads to constitutive activation of these MAP-kinase cascades, driving AP-1 activity.
Impact:
- Provides insights into the molecular mechanisms of oncogene-induced cell transformation.
- Highlights the role of MAP-kinase pathways in regulating cell proliferation.
- Identifies potential targets for therapeutic intervention in cancers driven by Ras pathway activation.