Nanyue Chen

ERKs and p38 Kinase Phosphorylate p53 Protein at Serine 15 in Response to UV Radiation

Phosphorylation of the p53 tumor suppressor protein is likely to play an important role in regulating its activity. Serine 15 phosphorylation of p53 leads to a stabilization of p53 by reducing its interaction with murine double minute 2, a negative regulatory partner. Recently, p53 was reported to be activated and phosphorylated at serine 15 following UV radiation. However, the signaling pathway that mediates UV-induced phosphorylation is less well characterized. Here, we provide evidence that UVB-induced phosphorylation of p53 at serine 15 is mediated directly by ERKs and p38 kinase. We find that in a mouse JB6 epidermal cell line, ERKs and p38 kinase form a complex with p53 following UVB radiation. Inhibition of ERKs or p38 kinase activity by the use of a dominant negative mutant of ERK2 or p38 kinase or their respective specific inhibitor, PD98059 or SB202190, results in abrogation of UVB-induced phosphorylation of p53 at serine 15. Strikingly, incubation of UVB-activated ERKs or p38 kinase immunoprecipitated complex with exogenous p53 shows serine 15 phosphorylation of both exogenous and co-precipitated endogenous p53 protein. Additionally, active recombinant ERK1/2 and p38 kinase but not JNKs are also able to phosphorylate p53 at serine 15 in vitro. Furthermore, pretreatment of cells with PD98059 or SB202190 blocks p53-dependent transcription activity but increases the level of p53 co-precipitated murine double minute. These results strongly suggest that both ERKs and p38 kinase have a direct role in UVB-induced phosphorylation of p53 at serine 15 in vivo.

Translocation of Protein Kinase Cε and Protein Kinase Cδ to Membrane Is Required for Ultraviolet B-induced Activation of Mitogen-activated Protein Kinases and Apoptosis

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCε and PKCδ, but not PKCα, from cytosol to membrane. Dominant negative mutant (DNM) PKCε or PKCδ inhibited UVB-induced translocation of PKCε and PKCδ, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCε and PKCδ, whereas the DNM of PKCα was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCδ, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCα, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 μm) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 μm), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCδ and PKCε, but not PKCα, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.

Cannabinoid-receptor-independent cell signalling by N-acylethanolamines

Anandamide and other polyunsaturated N-acylethanolamines (NAEs) exert biological activity by binding to cannabinoid receptors. These receptors are linked to G(i/o) proteins and their activation leads to extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAP kinase) activation, inhibition of cAMP-dependent signalling and complex changes in the expression of various genes. Saturated and monounsaturated NAEs cannot bind to cannabinoid receptors and may thus mediate cell signalling through other targets. Here we report that both saturated/monounsaturated NAEs and anandamide (20:4(n-6) NAE) stimulate cannabinoid-receptor-independent ERK phosphorylation and activator protein-1 (AP-1)-dependent transcriptional activity in mouse epidermal JB6 cells. Using a clone of JB6 P(+) cells with an AP-1 collagen-luciferase reporter construct, we found that 16:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n-6) NAEs stimulated AP-1-dependent transcriptional activity up to 2-fold, with maximal stimulation at approx. 10-15 microM. Higher NAE concentrations had toxic effects mediated by alterations in mitochondrial energy metabolism. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 signalling pathways, because all NAEs stimulated ERK1/ERK2 phosphorylation without having any effect on JNK or p38 kinases. Also, overexpression of dominant negative ERK1/ERK2 kinases completely abolished NAE-induced AP-1 activation. In contrast with 18:1(n-9) NAE and anandamide, the cannabinoid receptor agonist WIN 55,212-2 did not stimulate AP-1 activity and inhibited ERK phosphorylation. The NAE-mediated effects were not attenuated by pertussis toxin and appeared to be NAE-specific, as a close structural analogue, oleyl alcohol, failed to induce ERK phosphorylation. The data support our hypothesis that the major saturated and monounsaturated NAEs are signalling molecules acting through intracellular targets without participation of cannabinoid receptors.

Inhibition of atypical PKC blocks ultraviolet-induced AP-1 activation by specifically inhibiting ERKs activation.

Since ultraviolet (UV) radiation is a major etiologic factor in the development of human skin cancers, investigating the signal transduction pathways initiated by UV radiation may help with the understanding of the molecular mechanisms of UV‐induced carcinogenesis. Our previous studies demonstrated that UV‐induced activator protein‐1 (AP‐1) activation is blocked by dominant negative atypical PKCs (aPKCs). Here we investigated the role of aPKC in UV‐induced activation of mitogen activated protein (MAP) kinase family members which are considered to be the mediators of AP‐1 activation. We found that UV radiation led to translocation of protein kinase C (PKC) ζ and activation of MAP kinase family members as well as an increase of AP‐1–dependent transcription activation at the same dose range. Pretreatment of cells or mouse skin with antisense oligonucleotides of PKCζ impaired UV‐induced activation of AP‐1 in JB6 cells as well as in AP‐1–luciferase transgenic mice. It also inhibited UV‐induced activation of ERKs but not of JNK and p38 kinases in JB6 cells. In contrast, no significant inhibition of AP‐1 activation and MAP kinase activation were observed in cells treated with sense oligonucleotides of PKCζ. Furthermore, overexpression of a dominant negative mutant of PKCλ/ι specifically inhibited activation of extracellular signal–regulated protein kinases (ERKs) but not of c‐jun N‐terminal kinases (JNKs) nor p38 kinases induced by UV radiation. These results demonstrated that inhibition of aPKC impairs UV‐induced AP‐1 activation via suppression of ERKs activation but not of JNKs or p38 kinase activation. Mol. Carcinog. 27:65–75, 2000.

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.

Proteinase inhibitors I and II from potatoes block UVB-induced AP-1 activity by regulating the AP-1 protein compositional patterns in JB6 cells

Proteinase inhibitor I (Inh I) and proteinase inhibitor II (Inh II) from potato tubers are effective proteinase inhibitors of chymotrypsin and trypsin. Inh I and Inh II were shown to suppress irradiation-induced transformation in mouse embryo fibroblasts suggesting that they possess anticarcinogenic characteristics. We have previously demonstrated that Inh I and Inh II could effectively block UV irradiation-induced activation of transcription activator protein 1 (AP-1) in mouse JB6 epidermal cells, which mechanistically may explain their anticarcinogenic actions. In the present study, we investigated the effects of Inh I and Inh II on the expression and composition pattern of the AP-1 complex following stimulation by UV B (UVB) irradiation in the JB6 model. We found that Inh I and Inh II specifically inhibited UVB-induced AP-1, but not NFκB, activity in JB6 cells. Both Inh I and Inh II up-regulated AP-1 constituent proteins, JunD and Fra-2, and suppressed c-Jun and c-Fos expression and composition in bound AP-1 in response to UVB stimulation. This regulation of the AP-1 protein compositional pattern in response to Inh I or Inh II may be critical for the inhibition of UVB-induced AP-1 activity by these agents found in potatoes.

Inositol hexaphosphate inhibits ultraviolet B–induced signal transduction

Inositol hexaphosphate (InsP6) has an effective anticancer action in many experimental models in vivo and in vitro. Ultraviolet B (UVB) radiation is believed to be responsible for many of the carcinogenic effects related to sun exposure, and alteration in UVB‐induced signal transduction is associated with UVB‐induced carcinogenesis. Here we report the effects of InsP6 on UVB‐induced signal transduction. InsP6 strongly blocked UVB‐induced activator protein‐1 (AP‐1) and NF‐κB transcriptional activities in a dose‐dependent manner. InsP6 also suppressed UVB‐induced AP‐1 and nuclear factor κB (NF‐κB) DNA binding activities and inhibited UVB‐induced phosphorylation of extracellular signal‐regulated protein kinases (Erks) and c‐Jun NH2‐terminal kinases (JNKs). Phosphorylation of p38 kinases was not affected. InsP6 also blocked UVB‐induced phosphorylation of IκB‐α, which is known to result in the inhibition of NF‐κB transcriptional activity. InsP6 does not block UVB‐induced phosphotidylinositol‐3′ (PI‐3) kinase activity, suggesting that the inhibition of UVB‐induced AP‐1 and NF‐κB activities by InsP6 is not mediated through PI‐3 kinase. Because AP‐1 and NF‐κB are important nuclear transcription factors that are related to tumor promotion, our work suggests that InsP6 prevents UVB‐induced carcinogenesis by inhibiting AP‐1 and NF‐κB transcription activities.