Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation

Mitogen- and stress-activated protein kinases, MSK1 and the closely related isoform MSK2, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38 MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. Here we report that the CK2 protein kinase, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue Ser³²⁴ and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at Ser²⁷⁶ in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV radiation led to the activation of NF-κB-responsive gene expression in MDA-MB-231 cells and induced p65 transactivation capacity that was dependent on MSK2, MSK2 residue Ser³²⁴, and p65-Ser²⁷⁶. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at Ser²⁷⁶ that positively regulates NF-κB activity in MDA-MB-231 cells.     

The UVB-Stimulated Expression of Transglutaminase 1 Is Mediated Predominantly via the NFκB Signaling Pathway: New Evidence of Its Significant Attenuation through the Specific Interruption of the p38/MSK1/NFκBp65 Ser276 Axis

The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis.     

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.     

Herpes simplex virus type 1 ICP27-dependent activation of NF-kappaB

The ability of herpes simplex virus type 1 (HSV-1) to activate NF-kappaB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-kappaB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IkappaBalpha and IkappaBbeta. In this study, we identified loss of IkappaBalpha as a marker of NF-kappaB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IkappaBalpha loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IkappaBalpha loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-kappaB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-kappaB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IkappaBalpha and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-kappaB activation during HSV infection, highlighting the important role that NF-kappaB may play in infection.     

Acute alcohol exposure exerts anti-inflammatory effects by inhibiting IkappaB kinase activity and p65 phosphorylation in human monocytes

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.