p38/RK is essential for stress-induced nuclear responses: JNK/SAPKs and c-Jun/ATF-2 phosphorylation are insufficient.

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes (reviewed in [1]) are differentially activated in mammalian cells by various stimuli, which elicit induction of immediate-early (IE) genes, such as c-fos and c-jun (reviewed in [1-3]), as well as phosphorylation of histone H3 [4] and HMG-14 [5]. Anisomycin and UV radiation have been suggested to induce c-fos and c-jun transcription via JNK/SAPK-mediated phosphorylation of TCF (ternary complex factor), for c-fos induction [6-8], and c-Jun and/or ATF-2 for c-jun induction [9-11] [12,13]. We report here that anisomycin and ultraviolet radiation (UV) activate MAP kinase kinase-6 (MKK6) [14,15], p38/RK [16] [17,18] and MAPKAP kinase-2 (MAPKAP K-2) [17-19]. By using the p38/RK inhibitor SB 203580 [20,21], we show that activation of p38/RK and/or its downstream effectors are essential for anisomycin- and UV-stimulated c-fos/c-jun induction and histone H3/HMG-14 phosphorylation, whereas JNK/SAPK activation and phosphorylation of c-Jun and ATF-2 are insufficient for these responses.

Use of reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene columns for the rapid separation and purification of acid-soluble nuclear proteins.

The suitability of polystyrene-divinylbenzene reversed-phase HPLC columns for rapid separation and purification of acid-soluble nuclear proteins was evaluated. We used a polystyrene-divinylbenzene reversed-phase HPLC column (PLRP-S) for purification of nuclear proteins extracted with 0.3 M HCl or 5% HClO4. We are able to obtain electrophoretically pure fractions for a number of nuclear proteins including HMG14, HMG17 and variants of histone H3. The identity of proteins in these fractions was confirmed by immunochemical analysis, protein sequencing, mass spectrometry and migration on two-dimensional polyacrylamide gel electrophoresis. These methods do not require special preparation of the sample and are quicker than similar published methods.

Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors.

When quiescent cells are stimulated with growth factors, phorbol esters, okadaic acid, or protein synthesis inhibitors, the early-response genes, which include c-fos and c-jun, are rapidly induced. The earliest growth factor- and phorbol ester-stimulated nuclear signaling events concomitant with proto-oncogene induction are the rapid phosphorylation of two chromatin-associated proteins, pp33 and pp15. We show here that the tumor promoter okadaic acid, which inhibits protein phosphatases 1 and 2A, and the protein synthesis inhibitors anisomycin and cycloheximide also stimulate pp33 and pp15 phosphorylation. Using transcriptional inhibitors, we show that this response is not a consequence of early gene induction. By peptide mapping and microsequencing, chromatin-associated pp15 is identified as histone H3. Upon stimulation, histone H3 is rapidly phosphorylated on serine residues within its highly charged, basic N-terminal domain. Thus, these diverse agents elicit a common early nuclear signal modulating nucleosomal structure or function, potentially contributing to conformational regulation of proto-oncogene induction.

A mitogen- and anisomycin-stimulated kinase phosphorylates HMG-14 in its basic amino-terminal domain in vivo and on isolated mononucleosomes.

The rapid, transient induction of 80-100 immediate-early (IE) genes upon mitogenic stimulation occurs irrespective of protein synthesis and is mediated by modification of existing proteins. Two mechanisms, not mutually exclusive, involving modification either of sequence-specific transcription factors or of structural chromatin proteins primed by pre-association with responsive effectors are conceivable. Here, we show that upon IE gene induction, the non-histone high-mobility-group protein HMG-14, but not the related protein HMG-17, becomes serine phosphorylated in its basic, amino-terminal region close to where it binds nucleosomal DNA. Phosphorylation, normally transient, occurs independent of transcription and is quantitative and prolonged during superinduction. Brief micrococcal nuclease digestion substantially releases HMG-14 from nuclei in the mononucleosome-bound state. Finally, mononucleosomes prepared from mitogen-stimulated, but not control, cells contain a mitogen-activated kinase that phosphorylates HMG-14 in vitro on the same site(s) as in intact cells. The association of HMG-14 and its mitogen-activated kinase with nuclease-sensitive mononucleosomes has implications for models of mitogen-stimulated IE gene induction.

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.

Mitogen-stimulated phosphorylation of histone H3 is targeted to a small hyperacetylation-sensitive fraction.

Diverse agents, including growth factors and phorbol esters, induce rapid transcriptional activation of a subset of immediate-early (IE) genes that include the protooncogenes c-fos and c-jun. Among the earliest nuclear signaling events concomitant with IE gene activation is the phosphorylation of nucleosomal histone H3 in its basically charged N-terminal tail. This highly conserved domain is also subject to reversible posttranslational acetylation at specific lysine residues, a process implicated in transcriptional regulation. We show here that H3 phosphorylation associated with G0-G1 transition affects only a small fraction of this histone in the nucleus. Moreover, this fraction is biochemically distinct from bulk H3 in being extremely sensitive to sodium butyrate-induced hyperacetylation. However, acetylation itself does not predispose H3 to phosphorylation, nor does phosphorylation predispose H3 to enhanced acetylation. Further, selectivity is not based on preferential modification of particular histone H3 subtypes. Thus, the mitogen-regulated kinase that phosphorylates histone H3 is restricted to a small subset of nucleosomes that is especially susceptible to hyperacetylation.

The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase.

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, we show 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 (i) it is activated by both ERK and p38 MAPKs; (ii) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (iii) its activity towards 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.