Light signaling and UV-B-mediated plant growth regulation.

Light plays an important role in plants' growth and development throughout their life cycle. Plants alter their morphological features in response to light cues of varying intensity and quality. Dedicated photoreceptors help plants to perceive light signals of different wavelengths. Activated photoreceptors stimulate the downstream signaling cascades that lead to extensive gene expression changes responsible for physiological and developmental responses. Proteins such as ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) act as important factors which modulate light-regulated gene expression, especially during seedling development. These factors function as central regulatory intermediates not only in red, far-red, and blue light pathways but also in the UV-B signaling pathway. UV-B radiation makes up only a minor fraction of sunlight, yet it imparts many positive and negative effects on plant growth. Studies on UV-B perception, signaling, and response in plants has considerably surged in recent times. Plants have developed different strategies to use UV-B as a developmental cue as well as to withstand high doses of UV-B radiation. Plants' responses to UV-B are an integration of its cross-talks with both environmental factors and phytohormones. This review outlines the current developments in light signaling with a major focus on UV-B-mediated plant growth regulation.

Tunable light and drug induced depletion of target proteins.

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.

High expression of RAD18 in glioma induces radiotherapy resistance via down-regulating P53 expression.

As a key regulator of DNA translesion synthesis (TLS) pathway, RAD18 is reported to be abnormally expressed in many kinds of cancers. In glioma, RAD18 was overexpressed in the primary and recurrent glioblastoma multiforme specimens, and its overexpression weakened ionizing radiation-induced apoptosis in glioma A172 cells. Moreover, A172 cells with mutational P53 also showed enhanced radiation resistance. And RAD18 activation induced by cyclin-dependent kinase 2 (CDK2) was repressed by P53. However, whether P53 involves in RAD18-induced radiation resistance remains unknown. Therefore, this study was conducted to explore the effects and mechanism of RAD18 in the radiation resistance of glioma and study P53 role in this process. Results showed that, RAD18 expression was obviously elevated in glioma tissues and cell lines such as U251, SHG-44, A172, U-87 MG and U-118 MG as compared with the normal brain tissues and neuroglia cells. Up-regulation of RAD18 in U-118 MG and A172 cells with lentivirus infection significantly increased cell growth and inhibited cell apoptosis, determined by CCK-8 and flow cytometry technologies. Besides, RAD18 overexpression enhanced cell growth and inhibited cell apoptosis after U-118 MG or A172 cells were irradiated at a dose of 4 Gy. On the contrary, silencing of endogenous RAD18 sensitized U-118 MG and A172 cells to radiation. Moreover, RAD18 and P53 proteins were co-located in the nucleus, and up-regulation of RAD18 decreased the expression of P53 protein and facilitated its nuclear export. Furthermore, cell growth promotion and cell apoptosis inhibition induced by RAD18 up-regulation were impaired when P53 expression was up-regulated under radiation condition. In a word, this study clarifies that RAD18 functions as a promoter in glioma progression and reduces glioma cells' sensibility to radiation through down-regulating P53, which provides new strategies to overcome the radiation resistance in glioma.

Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)2(SPA1)2] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440-669 kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1L105A (which forms homodimers) and COP1L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440-669 kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.

Regulation of the ring finger E3 ligase Siah2 by p38 MAPK.

The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.

DNA damage-induced BARD1 phosphorylation is critical for the inhibition of messenger RNA processing by BRCA1/BARD1 complex.

BRCA1-associated RING domain protein BARD1, along with its heterodimeric partner BRCA1, plays important roles in cellular response to DNA damage. Immediate cellular response to genotoxic stress is mediated by a family of phosphoinositide 3-kinase-related protein kinases, such as ataxia-telangiectasia mutated (ATM), ATM and Rad3-related, and DNA-dependent protein kinase. ATM-mediated phosphorylation of BRCA1 enhances the DNA damage checkpoint functions of BRCA1, but how BARD1 is regulated during DNA damage signaling has not been examined. Here, we report that BARD1 undergoes phosphorylation upon ionizing radiation or UV radiation and identify Thr(714) as the in vivo BARD1 phosphorylation site. Importantly, DNA damage functions of BARD1 (i.e., inhibition of pre-mRNA polyadenylation and degradation of RNA polymerase II) are abrogated in T714A and T734A mutants. Our findings suggest that phosphorylation of BARD1 is critical for the DNA damage functions of the BRCA1/BARD1 complex.