Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene have no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) are stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor EGF). TPA and EGF induce the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this is abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 is barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation is similar, indicating that the MSK1 'knockout' does not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells (Arthur, 2000).
The 90 kDa ribosomal S6 kinase-2 (RSK2; see Drosophila S6kII) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Ser386 in the hydrophobic motif of RSK2 has been identified as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induces recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant show no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induces autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide [RSK2(373-396)] increases PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, are constitutively active in vivo and phosphorylate histone H3. These results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2 (Frodin, 2000).
The nucleosomal response refers to the rapid phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6 that occurs concomitantly with immediate-early (IE) gene induction in response to a wide variety of stimuli. Using antibodies against the phosphorylated residues, it has been shown that H3 and HMG-14 phosphorylation is mediated via different MAP kinase (MAPK) cascades, depending on the stimulus. The nucleosomal response elicited by TPA is ERK-dependent, whereas that elicited by anisomycin is p38 MAPK-dependent. In intact cells, the nucleosomal response can be selectively inhibited using the protein kinase inhibitor H89. MAPK activation and phosphorylation of transcription factors are largely unaffected by H89, whereas induction of IE genes is inhibited and its characteristics markedly altered. MSK1 is considered the most likely kinase to mediate this response because (1) it is activated by both ERK and p38 MAPKs; (2) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (3) its activity toward H3/HMG-14 is uniquely sensitive to H89 inhibition. Thus, the nucleosomal response is an invariable consequence of ERK and p38 but not JNK/SAPK activation, and MSK1 potentially provides a link to complete the circuit between cell surface and nucleosome (Thomson, 1999).
The fact that ERK and p38-mediated signaling culminates in a common nucleosomal response suggests that there may be common nuclear effectors. MAP kinases themselves can be ruled out because the sites of phosphorylation are not proline directed; thus, kinases further downstream are implicated. Several downstream effector kinases have been identified. Some, such as RSKs, are activated solely by ERKs, while others such as MAPKAP kinase 2 lie exclusively downstream of p38. However, two effector kinases, MNK1/2 and MSK1/2, can be activated by either ERK or p38; these represent points of convergence for signals from both pathways. Of these, MSK1 is reported to be localized in the nucleus, prompting an investigation to see if it might be responsible for phosphorylating histone H3 and HMG-14. These data show that MSK1 is an excellent candidate for mediating the nucleosomal response. (1) It is a nuclear kinase activated by both ERK and p38 MAP kinase pathways in C3H 10T1/2 cells. MSK1 is not activatable by JNK/SAPKs, in agreement with the observation that JNK/SAPK activation is insufficient for H3/HMG-14 phosphorylation. (2) It can efficiently phosphorylate HMG-14 and H3 substrates in vitro on physiologically relevant residues; it is much more efficient than RSK1 or 2 or MNK1 or 2. (3) H3 and HMG-14 phosphorylation in C3H 10T1/2 cells is highly selectively inhibited by H89 and the point of inhibition lies downstream of the MAP kinases; MSK1 satisfies this criterion and is inhibited in vitro by H89 at concentrations that inhibit the nucleosomal response in vivo. Most importantly, H89 does not significantly inhibit the activity of RSK1 and RSK2. This observation is important as it has been shown previously that RSKs can phosphorylate histone H3 in vitro and RSKs have been proposed as a possible mitogen-stimulated H3 kinase in vivo. The demonstration here that RSKs are much less efficient than MSK1 at phosphorylating H3 and HMG-14, that RSKs are insensitive to inhibition with H89 and that RSKs do not lie downstream of p38 argues strongly that the RSKs are unlikely to be the physiologically relevant H3/HMG-14 kinases in these cells, at least in response to stress-related stimuli. Finally, it is worth noting that while it inhibits MSK1 activity in vitro, H89 does not inhibit its activation in intact cells; in fact, an increase in TPA or anisomycin-stimulated state of activation of MSK1 is seen upon H89 treatment, alluding to negative feedback loops within these pathways. The fact that activation of MSK1 in vivo is not inhibited by H89, whereas its kinase activity towards histone H3 and HMG-14 is inhibited by this compound strongly suggests that inhibition of the nucleosomal response in intact cells occurs because H89 acts directly on activated MSK1, blocking its ability to phosphorylate histone H3 and HMG-14 (Thomson, 1999).
The process of IE gene induction involves interplay between differentially activated MAP kinase cascades, multiple transcription factor phosphorylation events and potentially also the phosphorylation and acetylation of histones. Part of this process includes the establishment of initiation complexes at the relevant promoters, which requires activation of transcription factors occupying upstream regulatory elements. It is the phosphorylation of these factors that appears crucial to triggering gene induction; this also affords a model by which distinct enzymatic activities required to execute chromatin modifications may be brought together at IE gene promoters. (1) Active ERK, JNK/SAPK and p38 MAP kinases must co-locate with DNA-bound transcription factors in order to phosphorylate them; there is considerable evidence of transcription factors being able to form stable complexes with MAP kinases. (2) MAP kinases are themselves known to bind avidly to some downstream effector kinases; in fact, MNKs, PRAK and MAPKAP kinase 3 have all been cloned as MAP kinase-binding proteins using two-hybrid screens. It is conceivable therefore that MAP kinases may convey downstream effector kinases to specific promoters. This would not apply for MSK1, which is reported to be a nuclear kinase. In this case, MSK1 may be pre-associated with specific promoters and only require arrival of its upstream kinase for activation. Both these models provide a mechanism for specific IE gene promoter-directed targeting of histone H3 and HMG-14 phosphorylation. (3) These transcription factors can themselves recruit coactivator complexes, which include HATs such as p300/CBP and pCAF, providing a mechanism by which histone acetylation may also be targeted to nucleosomes associated with these promoters. Elk-1 and c-Jun are both capable of functioning in this way and in the case of c-Jun, phosphorylation by JNK/SAPKs is reported to enhance CBP-binding. An implication of this type of model where two distinct histone-modifying activities coalesce around the same promoter is that acetylation and phosphorylation might be targeted to a common subset of H3 molecules; this is in fact exactly what is observed experimentally. Formaldehyde cross-linking and chromatin immunoprecipitation (CHIP) assays are being used with phospho-specific antibodies in an attempt to investigate the protein and DNA associated with these complexes. These studies show clearly that DNA encoding IE genes is co-immunoprecipitable from stimulated cells using antibodies against modified histone H3 and HMG-14, and proves for the first time that the nucleosomal response is targeted to chromatin associated with IE genes (Thomson, 1999).
Correlations between the nucleosomal response and IE gene expression under diverse conditions of induction, superinduction and inhibition strongly suggest that the two processes are linked mechanistically. By analogy with transcription factors, the phosphoepitope on histone H3 and HMG-14 may provide binding sites for recruitment of coactivators such as HATs or chromatin remodelling complexes. Alternatively, as suggested for acetylation of histone tails, phosphorylation may mediate a change in nucleosome and chromatin accessibility that aids transcription. IE genes show highly characteristic and reproducible patterns of induction with regard to the precise extent and duration of expression in response to different stimuli. In the presence of H89, when histone H3 and HMG-14 phosphorylation is blocked, IE gene induction is both inhibited and its pattern of expression altered, the reduced mRNA accumulation being more prolonged and observable at later time points. In agreement with the fact that MAP kinase activation and transcription factor phosphorylation are unaffected by H89, this shows that signals continue to arrive at these genes, but that the efficiency of their expression is inhibited. The argument that inhibition of IE gene induction by H89 is causal to the inhibited nucleosomal response is not tenable because the latter remains unaffected in the complete absence of transcription. Thus, although the nucleosomal response may not be obligate for IE gene induction in the way that transcription factor phosphorylation is, it may participate in quantitatively influencing the rate of expression of these genes. The products of the fos and jun family of proto-oncogenes homo- and hetero-dimerize to form the AP-1 complex, and the precise amounts of each protein partner must determine the nature of AP-1 complexes that result. In this light, quantitative modulation of IE gene transcript levels by the nucleosomal response may play a role in ultimately influencing the nature of the AP-1 complexes created in response to the diverse stimuli that elicit this response (Thomson, 1999).
A novel mitogen- and stress-activated protein kinase (MSK1) has been identified that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB (Deak, 1998).
LPS stimulation of RAW264 macrophages triggers the activation of mitogen- and stress-activated protein kinases-1 and -2 (MSK1 and MSK2) and their putative substrates, the transcription factors cyclic AMP response element-binding protein (CREB) and activating transcription factor-1 (ATF1). The activation of MSK1/MSK2 is prevented by preincubating the cells with a combination of two drugs that suppress activation of the classical mitogen-activated protein kinase cascade and stress-activated protein kinase/p38, respectively, but inhibition is only partial in the presence of either inhibitor. The LPS-stimulated activation of CREB and ATF1, the transcription of the cyclooxygenase-2 (COX-2) and IL-1 beta genes (the promoters of which contain a cyclic AMP response element), and the induction of the COX-2 protein are prevented by the same drug combination, as well as by Ro 318220 or H89, potent inhibitors of MSK1/MSK2. Two other transcription factors, C/EBP beta and NF-kappa B, have been implicated in the transcription of the COX-2 gene. However, PD 98059 and/or SB 203580 do not prevent the LPS-induced increase in the level of the transcription factor C/EBP beta, and none of the four inhibitors used in this study prevent the activation of NF-kappa B. These results demonstrate that two different mitogen-activated protein kinase cascades are rate limiting for the LPS-induced activation of CREB/ATF1 and the transcription of the COX-2 and IL-1 beta genes. They also suggest that MSK1 and MSK2 may play a role in these processes and hence are potential targets for the development of novel antiinflammatory drugs (Caivano, 2000).
Exercise/contraction is a powerful stimulator of mitogen-activated protein (MAP) kinase cascades in skeletal muscle. Little is known regarding the physiological activation of enzymes downstream of MAP kinase. An investigation was carried out to see whether acute exercise results in activation of mitogen- and stress-activated kinases (MSK) 1 and 2, p90 ribosomal S6 kinase (p90rsk), and MAP kinase-activated protein kinase 2 (MAPKAPK2). Muscle biopsies were obtained from healthy volunteers before, during, and after 60 min one-leg cycle ergometry, from exercising and resting legs. MSK1 and MSK2 activities were increased 400%-500% and 200%-300%, respectively, in exercised muscle. A dramatic increase in activity of p90rsk (MAPKAPK1), and to a lesser extent MAPKAP2, was noted with exercise. MSK1, MSK2, p90rsk, and MAPKAP2 activities are sustained throughout exercise. Exercise-induced activation of these enzymes is limited to working muscle, indicating that local rather than systemic factors activate these signaling cascades. Thus physical exercise leads to activation of multiple enzymes downstream of MAP kinase (Krook, 2000).
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