A novel member of lymphocyte-specific protein tyrosine kinase protein identified in lamprey, Lampetra japonica.

Abstract

Lymphocyte-specific protein tyrosine kinase p56 (Lck) is a member of the Src family of non-receptor protein tyrosine kinase. Lck plays an important role in mediating T-cell receptor (TCR) signal transduction and the development, differentiation, proliferation, and activation of T-cells [1]. Lck contains N-terminus of myristylation sequence, a unique aminoterminus region, followed by Src homology 3 (SH3) and SH2 domains and a C-terminus of tyrosine kinase catalytic domain [2]. Lck can associate with the inner face of the plasma membrane through its myristoyl glycine and palmitoyl cysteines in the amino-terminus [3].Following the myristylation sequence, there is the unique region, a short region of 80 amino acids. This unique region is involved in the interaction of Lck with specific cellular proteins [4,5]. Downstream of this unique region are SH3 and SH2 domains which are involved in protein–protein interactions [6]. The tyrosine kinase domain is the catalytic domain of Lck catalyzing the transfer of the gamma-phosphate from ATP to tyrosine residues in proteins. The catalytic domain of human Lck contains a site of autophosphorylation (Tyr-394), which plays an important role in regulating the protein kinase activity [7]. Agnathans, represented by lamprey and hagfish, are the oldest vertebrates currently identified possessing the adaptive immune defenses [8]. A recent study of jawless vertebrate has provided a clue for the origin of adaptive immune defense. Though TCR and B-cell receptor system do not exist in jawless vertebrates, lamprey has been confirmed to possess an alternative immune system that could specifically recognize and respond to external pathogens [9]. The handling of lamprey (Lampetra japonica) and all experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University. Adult lampreys were purchased from Tongjiang section of the Heilongjiang River (Tongjiang, China) in December. Adult lampreys (200–220 g in weight) were divided into two groups (20 animals per group); one group of animals was immunized with 0.1 mg of lipopolysaccharide (LPS) (Sigma-Aldrich, St Louis, USA) in 0.1 ml phosphate-buffered saline (PBS), and the control animals were injected with 0.1 ml PBS only. The animals were immunized at 8-day intervals by four intraperitoneal injections. Based on the expressed sequence tag analysis of the cDNA library which was constructed with lamprey lymphocyte-like cells in our laboratory, a Lck homolog was found using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI; http://www. ncbi.nlm.nih.gov/). Total RNA was isolated from lamprey lymphocyte-like cells [10] using RNAiso (TaKaRa Biotechnology, Dalian, China) reagent following the manufacturer instructions, and dissolved in Diethyl pyrocarbonate-treated water and stored at 2808C. First strand 30and 50-RACEcDNAs were synthesized from 5 mg of total RNA by Reverse Transcriptase M-MLV at 308C for 10 min, 428C for 30 min, 708C for 15 min, 958C for 5 min, 48C for 60 min with the 30-coding sequence primer and 50-coding sequence primer and Random 9-mers primer following the manufacturer instructions (TaKaRa Biotechnology). The 30and 50-end sequences of Lj-Lck were obtained by polymerase chain reaction (PCR) with outer primer, inner primer (TaKaRa Biotechnology), and specific primers (Supplementary Table S1). LA Taq DNA polymerase (TaKaRa Biotechnology) was used for amplification with the following cycling conditions: 948C for 3 min, followed by 40 amplification cycles at 948C for 30 s, 558C for 30 s, 728C for 2 min, and a final extension step at 728C for 10 min. Products were analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide. The target band of PCR product was isolated and purified, subcloned into Acta Biochim Biophys Sin 2014, 46: 820–825 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu066. Advance Access Publication 25 July 2014