When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.Histone H1 and the N-terminal tail of H3 have key roles in the folding and inter-association of the chromatin fiber (1-5). Modification of the N-and C-terminal tails of H1 by phosphorylation or the N-terminal tail of H3 by acetylation and/or phosphorylation could destabilize higher order chromatin structure (6, 7). Myc-and ras-transformed and Rb-deficient fibroblasts have a more decondensed chromatin structure than parental cells (8 -10). A general feature of these oncogenetransformed and Rb-deficient cells is increased H1 phosphorylation. H1 phosphorylation may relax chromatin by interfering with its action in chromatin folding and intermolecular fiberfiber interactions (3).Continuous stimulation of the Ras mitogen-activated protein kinase (MAPK) 1 signaling pathway in mouse fibroblasts transformed with oncogenes ras, fes, mos, and c-myc elevates the level of phosphorylated H1 (9, 11). Activation of the Ras-MAPK signaling pathway of quiescent fibroblasts treated with growth factors or phorbol esters results in the phosphorylation of H3 (12-14). Thus, persistent activation of the Ras-MAPK pathway in oncogene-transformed cells may also increase the level of phosphorylated H3, contributing to the destabilization of the higher order compaction of chromatin.The N-terminal domain of H3 can be phosphorylated on Ser-10 and/or Ser-28 (15, 16); however, the site of mitogeninduced phosphorylation remains unknown. Phosphorylation of H3 happens concurrently with the transcriptional activation of the immediate early response genes, e.g. c-fos (12, 13). However, inhibition of transcription does not prevent mitogen-activated H3 phosph...

The ERK group of mitogen-activated protein kinases (MAPKs) is essential for cell proliferation stimulated by mitogens, oncogenic ras and raf (ref. 1). All MAPKs are activated by MAP3K/MEK/MAPK core pathways and the Raf proto-oncoproteins, especially B-Raf, are ERK-specific MAP3Ks (refs 1-3). Mixed lineage kinase-3 (MLK3) is a MAP3K that was thought to be a cytokine-activated, and comparatively selective, regulator of the JNK group of MAPKs (refs 1, 4-6). Here we report that silencing of mlk3 by RNAi suppressed mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocked mitogen-stimulated phosphorylation of B-Raf at Thr 598 and Ser 601, a step required for B-Raf activation. Furthermore, silencing mlk3 prevented serum-stimulated cell proliferation and the proliferation of tumour cells bearing either oncogenic Ki-Ras or loss-of-function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumour cells containing activating B-raf or raf-1 mutations was unaffected by silencing mlk3. Our results define an unexpected role for MLK3 in mitogen regulation of B-Raf, ERK and cell proliferation.

We compared the nucleosomal organization, histone H1 subtypes, and histone H1 phosphorylated isoforms of ras-transformed and parental 10T1/2 mouse fibroblasts. In agreement with previous studies, we found that ras-transformed mouse fibroblasts have a less condensed chromatin structure than normal fibroblasts. ras-transformed and parental 10T1/2 cells had similar amounts of H1 subtypes, proteins that have a key role in the compaction of chromatin. However, labeling studies with 32P and Western blot experiments with an antiphosphorylated H1 antibody show that interphase ras-transformed cells have higher levels of phosphorylated H1 isoforms than parental cells. G1/S phase-arrested ras-transformed cells had higher amounts of phosphorylated H1 than G1/S phase-arrested parental cells. Mouse fibroblasts transformed with fes, mos, raf, myc, or constitutively active mitogen-activated protein (MAP) kinase kinase had increased levels of phosphorylated H1. These observations suggest that increased phosphorylation of H1 is one of the consequences of the persistent activation of the mitogen-activated protein kinase signal transduction pathway. Indirect immunofluorescent studies show that phosphorylated H1b is localized in centers of RNA splicing in the nucleus, suggesting that this modified H1 subtype is complexed to transcriptionally active chromatin.

The Ras 3 Raf 3 MEK1͞2 3 extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf͞Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a lossof-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin. extracellular signal-regulated kinase ͉ mitogen-activated protein kinase͞merlin M itogen-activated protein kinase (MAPK) kinase kinase (MAP3K) 3 MAPK͞extracellular signal-related kinase (ERK) kinase (MEK) 3 MAPK modules are evolutionarily conserved signaling networks that regulate numerous processes crucial to cell proliferation, survival, inflammation, and metabolism (1). The regulation of MAP3Ks is still poorly understood, especially with regard to how proximal elements that recruit different MAPKs integrate these pathways to produce an appropriate response. Mixed-lineage kinase 3 (MLK3) is one of a family of MAP3Ks, the MLKs, originally identified as specific regulators of the c-Jun N-terminal kinase (JNK) group of MAPKs (1, 2). MLK3 has been linked to TNF activation of JNK (3, 4), and, indeed, targeted disruption of mlk3 in mice impairs TNF recruitment of JNK (4). Other MAPK pathways are unaffected in mlk3 Ϫ/Ϫ mice (4). In contrast, we found that RNA interference (RNAi) silencing of mlk3 revealed, in multiple, diverse cell types, a broader function for MLK3. Thus, our results indicate that MLK3 is required for activation of ERK, JNK, and p38 MAPKs by mitogenic and proinflammatory stimuli (3). The reasons for the discrepancy between our findings and those for the mlk3 Ϫ/Ϫ mice are unclear and may reflect a compensatory redundancy among the different MLKs expressed in these mice, a phenomenon that presumably did not occur in our cultured cell models. Nevertheless, our findings point to a signal-integrating role for MLK3.Of particular interest, we uncovered a potential non-MAP3K role for MLK3 (3). B-Raf and the related Raf-1 are major MAP3Ks coupling Ras to ERKs 1 and 2 (ERK1͞2) and MAPK͞ ERK kinases 1 and 2 (MEK1͞2) (5). We observed that MLK3 was necessary for the signal-dependent phosphorylation of B-Raf...