UVA induces Ser381 phosphorylation of p90RSK/MAPKAP-K1 via ERK and JNK pathways

UVA exposure plays an important role in the etiology of skin cancer. The family of p90-kDa ribosomal S6 kinases (p90(RSK)/MAPKAP-K1) are activated via phosphorylation. In this study, results show that UVA-induced phosphorylation of p90(RSK) at Ser(381) through ERKs and JNKs, but not p38 kinase pathways. We provide evidence that UVA-induced p90(RSK) phosphorylation and kinase activity were time- and dose-dependent. Both PD98059 and a dominant negative mutant of ERK2 blocked ERKs and p90(RSK) Ser(381) phosphorylation, as well as p90(RSK) activity. A dominant negative mutant of p38 kinase blocked UVA-induced phosphorylation of p38 kinase, but had no effect on UVA-induced Ser(381) phosphorylation of p90(RSK) or kinase activity. UVA-induced p90(RSK) phosphorylation and kinase activity were markedly attenuated in JnK1(-/-) and JnK2(-/-) cells. A dominant negative mutant of JNK1 inhibited UVA-induced JNKs and p90(RSK) phosphorylation and kinase activity, but had no effect on ERKs phosphorylation. PD169316, a novel inhibitor of JNKs and p38 kinase, inhibited phosphorylation of p90(RSK), JNKs, and p38 kinase, but not ERKs. However, SB202190, a selective inhibitor of p38 kinase, had no effect on p90(RSK) or JNKs phosphorylation. Significantly, ERKs and JNKs, but not p38 kinase, immunoprecipitated with p90(RSK) when stimulated by UVA and p90(RSK) was a substrate for ERK2 and JNK2, but not p38 kinase. These data indicate clearly that p90(RSK) Ser(381) may be phosphorylated by activation of JNKs or ERKs, but not p38 kinase.     

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.     

MNK1, a new MAP kinase-activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates.

We have developed a novel expression screening method for identifying protein kinase substrates. In this method, a lambda phage cDNA expression library is screened by in situ, solid-phase phosphorylation using purified protein kinase and [gamma-32P]ATP. Screening a HeLa cDNA library with ERK1 MAP kinase yielded cDNAs of previously characterized ERK substrates, c-Myc and p90RSK, demonstrating the utility of this method for identifying physiological protein kinase substrates. A novel clone isolated in this screen, designated MNK1, encodes a protein-serine/threonine kinase, which is most similar to MAP kinase-activated protein kinase 2 (MAPKAP-K2), 3pK/MAPKAP-K3 and p90RSK. Bacterially expressed MNK1 was phosphorylated and activated in vitro by ERK1 and p38 MAP kinases but not by JNK/SAPK. Further, MNK1 was activated upon stimulation of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate, fetal calf serum, anisomycin, UV irradiation, tumor necrosis factor-alpha, interleukin-1beta, or osmotic shock, and the activation by these stimuli was differentially inhibited by the MEK inhibitor PD098059 or the p38 MAP kinase inhibitor SB202190. Together, these results indicate that MNK1 is a novel class of protein kinase that is activated through both the ERK and p38 MAP kinase signaling pathways.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

MAP kinases mediate UVB-induced phosphorylation of histone H3 at serine 28

Histone H3 phosphorylation is related closely to chromatin remodeling and chromosome condensation. H3 phosphorylation at serine 28 is coupled with mitotic chromosome condensation in diverse mammalian cell lines. However, the pathway that mediates phosphorylation of H3 at serine 28 is unknown. In the present study, ERK1, ERK2, or p38 kinase strongly phosphorylated H3 at serine 28 in vitro. JNK1 or JNK2 was able also to phosphorylate H3 at serine 28 in vitro but to a lesser degree. UVB irradiation markedly induced phosphorylation of H3 at serine 28 in JB6 Cl 41 cells. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently repressed UVB-induced H3 phosphorylation at serine 28. Expression of dominant negative mutant (DNM) ERK2 in JB6 Cl 41 cells totally blocked UVB-induced phosphorylation of H3 at serine 28. Additionally, DNM p38 kinase or DNM JNK1 partially blocked UVB-induced H3 phosphorylation at serine 28. Furthermore, UVB-induced H3 phosphorylation at serine 28 was inhibited in Jnk1(-/-) cells but not in Jnk2(-/-) cells. These results suggest that UVB-induced H3 phosphorylation at serine 28 may be mediated by mitogen-activated protein kinases.     

Kennedy's disease. Phosphorylation of the polyglutamine-expanded form of androgen receptor regulates its cleavage by caspase-3 and enhances cell death

X-linked spinal and bulbar muscular atrophy is a degenerative disease affecting motor neurons that is caused by polyglutamine (polyQ) expansion within the androgen receptor (AR). The polyQ-expanded form of AR is cytotoxic to cells, and proteolytic cleavage enhances cell death. The intracellular signaling pathways activated and/or required for cell death induced by the expanded form of AR (AR112) are unknown. We found that AR regulates mitogen-activated protein kinase (MAP kinase) pathways and, therefore, hypothesized that these pathway(s) may be required for AR112-induced cell death. The polyQ expansion in AR activates three MAP kinase pathways, causing increasing levels of phosphorylation of p44/42, p38, and SAPK/JNK MAP kinase. Inhibitors of either the JNK or p38 pathways had no effect on AR112-induced cell death, suggesting they are not required for polyQ-induced cell death. Strikingly, the MEK1/2 inhibitor, U0126, which selectively inhibits the p44/42 MAP kinase pathway, reduces AR112-stimulated cell death. The inhibition of the MEK1/2 pathway correlates directly with a change in phosphorylation state of the androgen receptor. Mutation of the MAP kinase consensus phosphorylation site in AR at serine 514 blocked AR-induced cell death and the generation of caspase-3-derived cleavage products. We propose a mechanism by which phosphorylation at serine 514 of AR enhances the ability of caspase-3 to cleave AR and generate cytotoxic polyQ fragments.     

Induction of EGFR-dependent and EGFR-independent signaling pathways by ultraviolet A irradiation

Most of the signal pathways involved in ultraviolet (UV)-induced skin carcinogenesis are thought to originate at plasma membrane receptors. However, UVA-induced signal transduction to downstream ribosomal protein S6 kinases, p70(S6K) and p90(RSK), is not well understood. In this report, we show that UVA stimulation of the epidermal growth factor receptor (EGFR) may lead to activation of p70(S6K)/p90(RSK) through phosphatidyl isositol (PI)-3 kinase and extracellular receptor-activated kinases (ERKs). Evidence is provided that phosphorylation and activation of p70(S6K)/p90(RSK) induced by UVA were prevented in Egfr(-/-) cells and were also markedly inhibited by the EGFR-specific tyrosine kinase inhibitors AG1478 and PD153035. Furthermore, EGFR tyrosine kinase inhibitors and EGFR deficiency significantly suppressed activation of PI-3 kinase and ERKs in regulating activation of p90(RSK)/p70(S6K) but had no effect on activation of c-Jun NH(2)-terminal kinases (JNKs) and p38 kinase in response to UVA. Thus, our results suggest that UVA-induced EGFR signaling may be required for activation of p90(RSK)/p70(S6K), PI-3 kinase, and ERKs but not JNKs or p38 kinase.     

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.     

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.     

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.     

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.     

Regulation of ultraviolet B-induced phosphorylation of histone H3 at serine 10 by Fyn kinase

Ultraviolet B (UVB) induces phosphorylation of histone H3 at serine 10, and mitogen-activated protein kinases are involved in this signal transduction pathway. Here we provide evidence that Fyn kinase, a member of the Src kinase family, is involved in the UVB-induced phosphorylation of histone H3 at serine 10. UVB distinctly increased Fyn kinase activity and phosphorylation. Fyn kinase inhibitors 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-d)pyramide and leflunomide, an Src kinase inhibitor, suppressed both UVB-induced phosphorylation of histone H3 at serine 10 and Fyn kinase activity and phosphorylation. UVB-induced phosphorylation of histone H3 at serine 10 was blocked by either a dominant-negative mutant of Fyn (DNM-Fyn) kinase or small interfering RNA of Fyn kinase. UVB-induced phosphorylation and activities of ERKs and protein kinase B/Akt were markedly inhibited by DNM-Fyn kinase. However, DNM-Fyn kinase did not inhibit UVB-induced phosphorylation of p38 MAPK or c-Jun N-terminal kinases. Active Fyn kinase phosphorylated histone H3 at serine 10 in vitro, and the phosphorylated Fyn kinase could translocate into the nucleus of HaCaT cells. These results indicate that Fyn kinase plays a key role in the UVB-induced phosphorylation of histone H3 at serine 10.     

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.     

Phosphorylation of 4E-BP1 is mediated by the p38/MSK1 pathway in response to UVB irradiation.

In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.     

Mitogen- and stress-activated protein kinase 1 mediates activation of Akt by ultraviolet B irradiation

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.