MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14

Cells respond to mitogenic or stress stimuli by the rapid induction of immediate-early (IE) genes, which occurs concomitantly with the phosphorylation of histone H3 and the high-mobility-group protein HMG-14. In mammalian cells this response is mediated via ERK and p38 MAP kinase pathways, but the identity of the downstream kinase that phosphorylates histone H3 has been contentious. One study, based on Coffin- Lowry cells defective in RSK2, reported that RSK2 was the histone H3 kinase, while a second study, based on the efficiency of RSKs and MSKs as in vitro histone H3 kinases, and their relative susceptibility to kinase inhibitors, suggested that MSKs were responsible. We show here that the histone H3 phosphorylation response is normal in Coffin-Lowry cells. Further more, we show that histone H3 and HMG-14 phosphorylation is severely reduced or abolished in mice lacking MSK1 and MSK2. We also show that, despite this, histone H3 acetylation is unimpaired in these cells and that IE genes can be induced, although at a reduced efficiency. We conclude that MSKs are the major kinases for histone H3 and HMG-14 in response to mitogenic and stress stimuli in fibroblasts.     

Glutamate induces histone H3 phosphorylation but not acetylation in striatal neurons: role of mitogen- and stress-activated kinase-1

Chromatin remodelling is thought to play a key role in gene regulation that underlies long-term synaptic plasticity and memory formation. The dynamic process of chromatin remodelling requires post-translational modifications of histones, a group of highly basic proteins that are tightly linked to DNA. In the present study, we investigated histone H3 modifications in response to glutamate stimulation leading to c-Fos and c-Jun induction in an in vitro model system of striatal neurons in culture. Intracellular signalling pathways implicated in these modifications were analysed. Histone H3 acetylation was strong in basal conditions and unmodified by glutamate treatment. By contrast, glutamate induced a strong phosphorylation of histone H3 that was inhibited by selective inhibitors of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, U0126 and SB203580, respectively. Blocking activation of mitogen- and stress-activated kinase 1 (MSK1), a kinase downstream ERK and p38 MAPK, by pharmacological approach or using striatal cells from MSK1 deficient mice, totally abolished H3 phosphorylation, as well as c-Fos and c-Jun induction. Chromatin immunoprecipitation assays confirmed increased levels of phosphorylated H3 at the c-jun promoter. Altogether, our data highlight the crucial role of MSK1 in the nucleosomal response necessary for gene induction in neuronal cells.     

Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38(MAPK) phosphorylation. Phosphorylation of ERK1/2 or p38(MAPK) was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38(MAPK) was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38(MAPK) inhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90(Rsk)), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90(Rsk) and MAPKAP-K2 most closely reflects ERK and p38(MAPK) stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38(MAPK). These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.     

Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation

The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, the phosphorylation of histone H3 and HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Here, we provide direct evidence that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes becomes doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

蛋白激酶MSK在表观遗传学调控中的作用及其生物学意义

丝裂原和应激激活的蛋白激酶(MSK)是一类核内丝/苏氨酸蛋白激酶,参与丝裂原激活蛋白激酶(MAPK)信号通路介导的下游基因转录调控和表观遗传学调控.首先,MSK是MAPK通路的下游媒介分子.在丝裂原或应激刺激下,p38或ERK激酶通过级联磷酸化激活MSK蛋白.然后,活化的MSK介导转录因子磷酸化活化和组蛋白H3的10位丝氨酸磷酸化.MSK介导的组蛋白H3磷酸化,可引发组蛋白乙酰化和甲基化修饰的动态变化,相互协同或拮抗,开放染色质结构,利于诱导型基因的表达.除组蛋白H3外,MSK直接磷酸化的下游底物还包括CREB、NF-κB等转录因子以及多个非转录相关蛋白.因此,MSK能在多层次调控基因表达和细胞功能,广泛参与肿瘤转化、炎症反应、神经突触可塑性以及心肌肥大等生物学事件.本文将简要介绍MSK蛋白的研究进展,探讨其在转录调控、表观遗传学修饰等生物学事件中的作用.     

Dynamic changes in histone H3 phosphoacetylation during early embryonic stem cell differentiation are directly mediated by mitogen- and stress-activated protein kinase 1 via activation of MAPK pathways

Embryonic stem (ES) cells are pluripotent cells capable of unlimited self-renewal and differentiation into the three embryonic germ layers under appropriate conditions. Mechanisms for control of the early period of differentiation, involving exit from the pluripotent state and lineage commitment, are not well understood. An emerging concept is that epigenetic histone modifications may play a role during this early period. We have found that upon differentiation of mouse ES cells by removal of the cytokine leukemia inhibitory factor, there is a global increase in coupled histone H3 phosphorylation (Ser-10)-acetylation (Lys-14) (H3 phosphoacetylation). We show that this occurs through activation of both the extracellular signal-regulated kinase (ERK) and p38 MAPK signaling pathways. Early ES cell differentiation is delayed using pharmacological inhibitors of the ERK and p38 pathways. One common point of convergence of these pathways is the activation of the mitogen- and stress-activated protein kinase 1 (MSK1). We show here that MSK1 is the critical mediator of differentiation-induced H3 phosphoacetylation using both the chemical inhibitor H89 and RNA interference. Interestingly, inhibition of H3 phosphoacetylation also alters gene expression during early differentiation. These results point to an important role for both epigenetic histone modifications and kinase pathways in modulating early ES differentiation.     

ERKs and p38 kinases mediate ultraviolet B-induced phosphorylation of histone H3 at serine 10

Histone H3 is the core protein of the nucleosome. Phosphorylation of H3 involves immediate early gene expression, chromatin remodeling, and chromosome condensation during mitosis. Very recently, Rsk2 or MSK1 kinase-mediated phosphorylation of H3 at serine 10 was reported. In the present study, we show that both ERKs and p38 kinase may mediate ultraviolet B-induced phosphorylation of H3 at serine 10. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently inhibited ultraviolet B-induced phosphorylation of H3. Phosphorylation of H3 was also inhibited in cells expressing dominant negative mutant (DNM) ERK2 and DNM p38 kinase. In contrast, no inhibition of H3 phosphorylation in Jnk1 or Jnk2 knockout cells (Jnk1(-/-) or Jnk2(-/-)) and cells expressing DNM JNK1 was observed. More importantly, incubation of active ERK2 or p38 kinase with H3 protein resulted in phosphorylation of H3 at serine 10 in vitro. These results suggest that ERK and p38 kinase are at least two important mediators of phosphorylation of H3 at serine 10.     

Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1.

N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.     

Docosahexaenoic acid inhibits UVB-induced activation of NF-κB and expression of COX-2 and NOX-4 in HR-1 hairless mouse skin by blocking MSK1 signaling

Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Docosahexaenoic acid (DHA), a representative ω-3 polyunsaturated fatty acid, has been reported to possess anti-inflammatory and chemopreventive properties. In the present study, we investigated the molecular mechanisms underlying the inhibitory effects of DHA on UVB-induced inflammation in mouse skin. Our study revealed that topical application of DHA prior to UVB irradiation attenuated the expression of cyclooxygenase-2 (COX-2) and NAD(P)H:oxidase-4 (NOX-4) in hairless mouse skin. DHA pretreatment also attenuated UVB-induced DNA binding of nuclear factor-kappaB (NF-κB) through the inhibition of phosphorylation of IκB kinase-α/β, phosphorylation and degradation of IκBα and nuclear translocation of p50 and p65. In addition, UVB-induced phosphorylation of p65 at the serine 276 residue was significantly inhibited by topical application of DHA. Irradiation with UVB induced phosphorylation of mitogen and stress-activated kinase-1 (MSK1), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, and all these events were attenuated by pretreatment with DHA. Blocking ERK and p38 MAP kinase signaling by U0126 and SB203580, respectively, diminished MSK1 phosphorylation in UVB-irradiated mouse skin. Pretreatment with H-89, a pharmacological inhibitor of MSK1, abrogated UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 in mouse skin. In conclusion, topically applied DHA inhibits the UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 by blocking the phosphorylation of MSK1, a kinase downstream of ERK and p38 MAP kinase, in hairless mouse skin.     

Endothelin-1 induces serine phosphorylation of the adaptor protein p66Shc and its association with 14-3-3 protein in glomerular mesangial cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells (GMC). In the current study, it is demonstrated that ET-1 treatment of GMC results in serine phosphorylation of the 66-kDa isoform of the adapter protein Shc (p66(Shc)). ET-1-induced serine phosphorylation of p66(Shc) requires activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling module and is efficiently inhibited by both a MAPK/ERK kinase (MEK)-selective inhibitor and adenovirus-mediated transfer of a dominant interfering MEK1 mutant. Furthermore, adenovirus-mediated transfer of a constitutively active MEK1 mutant was found to markedly increase p66(Shc) serine phosphorylation. Adenoviruses encoding constitutively active mutants of MAPK kinases 3 and 6 (upstream kinases of p38(MAPK)) and 7 (upstream kinase of c-Jun NH(2)-terminal kinase) failed to induce serine phosphorylation of this adaptor protein. Serine phosphorylation of p66(Shc) resulted in its association with the serine binding motif-containing protein 14-3-3. ET-1-induced phosphorylation of a serine encompassed in the 14-3-3 binding motif of p66(Shc) was confirmed in experiments employing anti-phospho-14-3-3 binding motif antibodies. These studies are the first to demonstrate that G protein-coupled receptors stimulate serine phosphorylation of p66(Shc) and the first to report the formation of a signaling complex between p66(Shc) and 14-3-3.     

Critical role of cAMP-response element-binding protein for angiotensin II-induced hypertrophy of vascular smooth muscle cells

We reported previously an important role of cyclic AMP-response element (CRE) for the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE-binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). AngII time dependently induced phosphorylation of CREB with a peak at 5 min. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor antagonist, suggesting that AngII type I receptor may mediate the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. Inhibition of epidermal growth factor-receptor by AG1478 suppressed the AngII-induced CREB phosphorylation as well as activation of ERK and p38MAPK. Overexpression of the dominant negative form of CREB by an adenovirus vector suppressed AngII-induced c-fos expression and incorporation of [(3)H]leucine to VSMC. These findings suggest that AngII may activate multiple signaling pathways involving two MAPK pathways and protein kinase A, all of which contribute to the activation of CREB. Transactivation of epidermal growth factor-receptor is also critical for AngII-induced CREB phosphorylation. Activation of CREB may be important for the regulation of gene expression and hypertrophy of VSMC induced by AngII.     

PGE2 inhibits apoptosis in human adenocarcinoma Caco-2 cell line through Ras-PI3K association and cAMP-dependent kinase A activation

PGE2 plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and PGE2 synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that PGE2 has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of PGE2 on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by PGE2. In addition, PGE2 is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of PGE2 on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer.