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Identification of physiological substrate for a newly identified serine/threonine/tyrosine protein kinase
thesisposted on 2017-03-22, 01:29 authored by Ho, Hooi Ling
Protein phosphorylation is a post-translational modification process in which an amino acid residue in a protein is covalently modified by addition of a phosphate group involving an enzyme known as protein kinase. Many studies demonstrated prominent roles of protein kinases in the regulation of cell differentiation, growth and development. Protein kinases are also implicated in different signal transduction pathways in the abiotic and biotic stresses. By identifying substrates of the protein kinases, wide assessment on how the intact cell regulates cell growth and responses to external stimuli could be disclosed. Thus, the main objective of the study is to identify in vivo physiological substrate for a newly isolated serine/threonine/tyrosine protein kinase from Arabidopsis thaliana (designated as AtSTYK) and thereafter to uncover the role of AtSTYK in plant system. AtSTYK gene was assessed for the protein expression study in yeast system. SDS-PAGE analysis revealed molecular weight of the oligohistidine fusion AtSTYK protein was 49 kDa, indicating the recombinant gene was expressed in eukaryotic system. Thereafter, Western blot confirmed protein expression of AtSTYK in yeast system. The growth profile of recombinant bacteria and yeast was also determined. The study was undertaken to assess growth behavior and stability of recombinant vectors in the hosts. Higher growth rate of wild type was observed compared to recombinant strains during logarithmic phase. Plasmid stability studies revealed AtSTYK gene was able to retain in recombinant hosts after prolonged culturing. PCR screening and nucleotide sequencing analyses proved AtSTYK was present and amplified in many generations without any nucleotide changes. What is the physiological substrate for this AtSTYK to transfer a phosphate group? To address this, protein-protein interaction via yeast two-hybrid system was used to detect positive interactors of AtSTYK. The results showed that OBERON1 (OBE1) with its paralog, OBERON2 (OBE2) genes were detected as the most frequent interacting partners for AtSTYK. Taken together, the research findings suggest that OBE1 and OBE2 genes encode for plant homeodomain (PHD) finger proteins could be phosphorylated by AtSTYK. These results suggest AtSTYK plays a putative role in the maintenance and establishment of apical meristems as well as preservation of stem cells population within the apical meristems region. The maintenance of meristems is achieved through an equilibrium attained between propagation of pluripotent stem cells occurred at the center and organogenesis initiated on the flanks of the meristems.