Figure 6.
Figure 6. B-Raf is required for MEK/ERK phosphorylation and ERK activation is required for differentiation in oligodendrocyte-enriched glial cell cultures. Immunoblot analysis of whole cell lysates (40 μg) from WT and B-Raf KO oligodendrocyte-enriched (A and C) or oligodendrocyte-depleted mixed glial cell cultures (B) left untreated or stimulated for 10 min with 100 ng/ml PDGF (A and B; 10 min), 20 ng/ml EGF (C), or 20 ng/ml FGF (C) before lysis. MEK/ERK phosphorylation is impaired in oligodendrocyte-containing but not oligodendrocyte-depleted cultures. In B, the first lane from the left is slightly underloaded. (D) ERK phosphorylation in oligodendrocyte cultures. Cells allowed to differentiate for 2 d and either left untreated or stimulated with 20 ng/ml FGF for the indicate times. Cells were colabeled with antibodies against pERK (green) and pan-tubulin (red). (E) MEK1 immunoprecipitates were prepared from 400 μg WT and B-Raf null (KO) oligodendrocyte-enriched glial cell cultures, and left untreated or stimulated with 100 ng/ml PDGF (for 10 min) before lysis and immunoblotting. C, isotype-matched irrelevant Ab control; MEK1 KO, MEK1 ips prepared from whole cell lysate of MEK1 KO MEFs. (F) ERK phosphorylation in cells of the oligodendrocyte lineage in situ. Brain sections were colabeled with antibodies against pERK (green) and βIV-tubulin (red). Cells were classified on the basis of their morphology as premyelinating oligodendrocytes (left and middle) and mature oligodendrocytes (right). Asterisks indicate autofluorescent erythrocytes. Bars, 30 μm. (G) A working model Raf/MEK/ERK complexes in differentiating oligodendrocytes. Soluble or axonal signals activate the ERK pathway in oligodendrocyte precursors. In WT cells, three complexes may be formed, two of which are B-Raf dependent: complex I, which comprises B-Raf, KSR, and MEK/ERK; and complex II, which consists of a B-Raf–Raf-1 heterodimer bound to KSR and MEK/ERK. In complex I, MEK is phosphorylated by B-Raf, whereas in complex II, the Raf heterodimer is the MEK kinase. Both complexes produce a strong, sustained ERK signal ultimately leading to oligodendrocyte differentiation. Complex III comprises Raf-1 and MEK/ERK, is B-Raf independent, and is the only complex found in B-Raf–deficient oligodendrocytes. This complex gives rise to a much weaker ERK signal, leading to defective oligodendrocyte differentiation.

B-Raf is required for MEK/ERK phosphorylation and ERK activation is required for differentiation in oligodendrocyte-enriched glial cell cultures. Immunoblot analysis of whole cell lysates (40 μg) from WT and B-Raf KO oligodendrocyte-enriched (A and C) or oligodendrocyte-depleted mixed glial cell cultures (B) left untreated or stimulated for 10 min with 100 ng/ml PDGF (A and B; 10 min), 20 ng/ml EGF (C), or 20 ng/ml FGF (C) before lysis. MEK/ERK phosphorylation is impaired in oligodendrocyte-containing but not oligodendrocyte-depleted cultures. In B, the first lane from the left is slightly underloaded. (D) ERK phosphorylation in oligodendrocyte cultures. Cells allowed to differentiate for 2 d and either left untreated or stimulated with 20 ng/ml FGF for the indicate times. Cells were colabeled with antibodies against pERK (green) and pan-tubulin (red). (E) MEK1 immunoprecipitates were prepared from 400 μg WT and B-Raf null (KO) oligodendrocyte-enriched glial cell cultures, and left untreated or stimulated with 100 ng/ml PDGF (for 10 min) before lysis and immunoblotting. C, isotype-matched irrelevant Ab control; MEK1 KO, MEK1 ips prepared from whole cell lysate of MEK1 KO MEFs. (F) ERK phosphorylation in cells of the oligodendrocyte lineage in situ. Brain sections were colabeled with antibodies against pERK (green) and βIV-tubulin (red). Cells were classified on the basis of their morphology as premyelinating oligodendrocytes (left and middle) and mature oligodendrocytes (right). Asterisks indicate autofluorescent erythrocytes. Bars, 30 μm. (G) A working model Raf/MEK/ERK complexes in differentiating oligodendrocytes. Soluble or axonal signals activate the ERK pathway in oligodendrocyte precursors. In WT cells, three complexes may be formed, two of which are B-Raf dependent: complex I, which comprises B-Raf, KSR, and MEK/ERK; and complex II, which consists of a B-Raf–Raf-1 heterodimer bound to KSR and MEK/ERK. In complex I, MEK is phosphorylated by B-Raf, whereas in complex II, the Raf heterodimer is the MEK kinase. Both complexes produce a strong, sustained ERK signal ultimately leading to oligodendrocyte differentiation. Complex III comprises Raf-1 and MEK/ERK, is B-Raf independent, and is the only complex found in B-Raf–deficient oligodendrocytes. This complex gives rise to a much weaker ERK signal, leading to defective oligodendrocyte differentiation.

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