Molecular cloning, Expression and Functional Analysis of Zebrafish PYK2 Gene

• Main Authors: Yi-Hsin Lin, 林宜欣
• Other Authors: Wen-Chang Chang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/58506727384551728366

碩士 === 國立臺灣大學 === 生化科學研究所 === 91 === PYK2, a member of the focal-adhesion kinase family, belongs to non-receptor tyrosine kinase that is sensitive to variations in intracelluar Ca2+ level and stressful stimuli. A recent study showed that PYK2 is able to tyrosine-phosphorylate glycogen synthase kinase 3 β (GSK 3β) both in vitro and in vivo, and thus could be a key regulator of this protein kinase. GSK3β is a well-established component of Wnt signaling pathway, which is essential for establishing the entire body pattern during embryonic development. Therefore it is essential to know whether such a regulatory mechanism also exists during embryonic development and to what extent it may affect in the GSK3β-mediated pathway. Until now, the deciphering of zebrafish genome has not been finished. In this study, we focused our research mainly on the molecular cloning of PYK2 gene and characterization of its sequence. Initially, we adopted zebrafish embryo at primordial stage as an experimental material and synthesized the first strand cDNA using RT-PCR. According to the published protein sequence of the highly-conserved kinase domain, we designed a set of degenerate primers and cloned a fragment of 837 bp in length by PCR. Finally, we obtained the full-length PYK2 cDNA containing 5’ and 3’ UTR by means of RACE. PYK2 cDNA has 3792 bp in length and encodes 1004 amino acid residues. The result from multiple sequence alignment among species indicates that PYK2 exhibits a high degree of conservation during evolution. The data of Southern blotting analysis has shown that PYK2 gene exists in more than one copy. In order to investigate its temporal and spatial expression pattern, we approached the issues by RT-PCR and Whole-mount in situ hybridization, respectively. In terms of temporal expression, we realize PYK2 is a maternally-derived gene; on the other hand, its expression is localized mainly on brain at embryonic stage and becomes ubiquitously distributed in the adult fish. Secondly, we subcloned PYK2 cDNA in full length (rPYK2) and cDNA encoding kinase domain (rPYK2-CD) into expression vectors and induced their expression in E. coli. The results have shown that both of them exist in the form of inclusion body. For the sake of solublizing them for further analysis, we conquered this problem by column refolding and obtained soluble rPYK2-CD successfully; in addition, we also harvested soluble protein product of PYK2 cDNA in full length (rPYK2-IVT-HA) by in vitro translation. rPYK2-CD was found phosphorylated prior to the addition of ATP while we conducted in vitro kinase assay. We demonstrated that rPYK2-CD was phosphorylated while expressed in E. coli. In order to get deeper insight into this unusual phenomenon, we treated rPYK2-CD with calf intestine phosphatase first. After purified by Ni-NTA affinity column, CIP-treated rPYK2-CD was then incubated with E.coli lysate. The intensity of phosphorylation seemed to recover after incubation. The outcome demonstrates that E. coli is capable of tyrosine-phosphorylating rPYK2-CD. From in vitro kinase assay, we know rPYK2-IVT-HA undergoes autophosphorylation upon adding ATP. However, CIP-treated rPYK2-CD was unable to phosphrylate itself. Incubating rPYK2-CD with Raytide as a substrate reveals that kinase domain alone is sufficient to exert kinase activity.