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A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf
S L Sutor1, B T Vroman, E A Armstrong
1Division of Oncology Research, Mayo Foundation, Rochester, Minnesota 55905, USA.
Abstract:
Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.
Insights
Interleukin-3 (IL-3) signaling activates the MEK-Erk pathway in myeloid cells via A-Raf, not c-Raf. This activation depends on phosphatidylinositol 3-kinase (PI3-K) signaling, highlighting distinct upstream regulation of Raf isoforms.
Area of Science:
- Cellular signaling pathways
- Hematopoiesis
- Molecular biology
Background:
- Cytokines orchestrate cell signaling through protein kinase cascades.
- Mitogen-activated protein/ERK kinase (MEK) and Erk are key kinases in growth factor-regulated proliferation and survival.
- Interleukin-3 (IL-3) is a critical cytokine for myeloid progenitor cell development.
Purpose of the Study:
- To elucidate the upstream signaling mechanisms regulating MEK-Erk activation by IL-3 in myeloid progenitor cells.
- To determine the specific role of Raf isoforms (c-Raf, A-Raf, B-Raf) in IL-3-induced MEK activation.
- To investigate the involvement of phosphatidylinositol 3-kinase (PI3-K) and cyclic AMP (cAMP) in these signaling pathways.
Main Methods:
- Pharmacological inhibition of PI3-K and manipulation of cAMP levels.
- Genetic approaches using dominant-negative mutants of Raf isoforms.
- Analysis of Raf and MEK activation in response to IL-3 stimulation.
Main Results:
- IL-3-induced MEK and Erk activation requires a PI3-K-generated signal.
- A-Raf, but not B-Raf or c-Raf, is activated by IL-3 in a PI3-K-dependent manner.
- Dominant-negative A-Raf, but not c-Raf, blocks IL-3-induced MEK activation, indicating A-Raf mediates the signaling cascade.
Conclusions:
- IL-3 signaling activates both c-Raf and A-Raf in hemopoietic cells.
- A-Raf is the critical mediator of IL-3-induced MEK-Erk activation, regulated by PI3-K.
- Differential regulation of Raf isoforms by upstream signaling pathways dictates downstream responses.