Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina) Lymphocytes

Abstract

Protein kinases (PKs) play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs), successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine) kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK) and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

PAH‐ and PCB‐induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina) PBMC

“…AY578791, Bozza and Atkinson, unpublished (IL-1); GenBank acc. #AY611616 (Fyn) and #AY611617 (Itk), Neale et al 2004]. IL-2 primers were derived from the nucleotide sequence from another phocid, the northern elephant seal (Mirounga angustirostris; GenBank acc.…”

Dose-response relationships in gene expression profiles in a harbor seal B lymphoma cell line exposed to 17α-ethinyl estradiol

“…Mitogen-activated protein kinase kinase 3 (MAPKK3) activates MAPK3, a protein involved in the regulation of cellular processes such as proliferation and differentiation in lymphocytes. 24 MAPKK3 mRNA was upregulated in response to 25,000 μg/L EE 2 + LPS exposure, but not when the mitogen LPS was not present (Figure 1A, Table 2). A change in the MAP kinase pathway could impact both B cell receptor or T cell receptor signaling and T cell development.…”