Publications of NIPGR Scientists

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Author: search.filters.author.Sinha, Alok KrishnaSubject: search.filters.subject.protein stability

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    Dynamic phosphorylation of miRNA biogenesis factor HYL1 by MPK3 involving nuclear–cytoplasmic shuttling and protein stability in Arabidopsis
    (MDPI AG, 2022) Bhagat, Prakash Kumar; Verma, Deepanjali; Singh, Kirti; Badmi, Raghuram; Sharma, Deepika; Sinha, Alok Krishna
    MicroRNAs (miRNAs) are one of the prime regulators of gene expression. The recruitment of hyponastic leaves 1 (HYL1), a double-stranded RNA binding protein also termed as DRB1, to the microprocessor complex is crucial for accurate primary-miRNA (pri-miRNA) processing and the accumulation of mature miRNA in Arabidopsis thaliana. In the present study, we investigated the role of the MAP kinase-mediated phosphorylation of AtHYL1 and its sub-cellular activity. AtMPK3 specifically phosphorylates AtHYL1 at the evolutionarily conserved serine-42 present at the Nterminal regions and plays an important role in its nuclear–cytosolic shuttling. Additionally, we identified that AtHYL1 is cleaved by trypsin-like proteases into an N-terminal fragment, which renders its subcellular activities. We, for the first time, report that the dimerization of AtHYL1 not only takes place in the nucleus, but also in the cytosol, and the C-terminal of AtHYL1 has a role in regulating its stability, as well as its subcellular localization. AtHYL1 is hyper-phosphorylated in mpk3 mutants, leading to higher stability and reduced degradation. Our data show that AtMPK3 is a negative regulator of AtHYL1 protein stability and that the AtMPK3-induced phosphorylation of AtHYL1 leads to its protein degradation.
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    Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana
    (Frontiers Media S.A., 2016) Sheikh, Arsheed H.; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok Krishna; Scheel, Dierk; Lee, Justin
    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense.