八肋游仆虫中心蛋白磷酸化位点的鉴定.docVIP

 Identification of Phosphorylation Sites on Ciliate Euplotes Octocarinatus Centrin# 5  10  Abstract:  Phosphorylation of centrin is associated with alterations in contractile fibers. To obtain insight into the structural basis for the functional effect of phosphorylation, Ciliate Euplotes octocarinatus centrin (EoCen) phosphorylation by protein kinase A (PKA) was produced and 15 20 25 30 35 40 characterized by 31P-NMR. In 50 mM Tris-HCl, 5.0 mM Mg2+ and 1.2 mM ATP, EoCen can be phosphorylated by protein kinase A (PKA). The 31P-NMR of PKA phosphorylating N-terminal domain of EoCen (N-EoCen) and C-terminal domain of EoCen (C-EoCen) suggested that there existed maybe phosphorylation site on the two half domains. Through insert initiator codon ATG before N-EoCen (N+1-EoCen) elimating the effect of Ser on expression vector, we identified that Ser166 at C-terminal domain was the sole phosphorylation site of PKA on EoCen. Phosphorylated EoCen (EoCenp) may bind with mimic peptide melittin. Key words: Centrin; Phosphorylation; Melittin 0 Introduction The microtubule-based cytoskeleton in eukaryotic cells is organized and regulated by microtubule organizing centers（MTOC）. The MTOC in animal cells is known as the centrosome, and the equivalent organelle in the yeast Saccharomyces cerevisiae is called the spindle pole body (SPB) [1-3]. The centrosome consists of many proteins, most of which have remained unidentified. Centrin (also known as caltractin) is one of the conserved centrosomal proteins. It is a small acid (~ 20 KD) calcium binding protein, which was initially identified as major components of Ca2+-sensitive contractile fibers in the flagellar apparatus of the unicellular green algae, such as Tetraselmis striata and Chlamydomonas reinhardtii [4, 5]. Subsequently, centrin was found from protozoa and yeast to plants and human [6, 7]. Genetic studies show that centrins are essential for signal transduction via phosphorylation or dephosphorylation, proper ce