The 70 kDa S6 kinase : enrichment and characterization of sea star homologue, and the role of p70S6K in the transformed keratinocyte cell line Pam212 in response to photodynamic therapy
Creators: name: Charlton, Lorin Ann
Publisher: name: University of British Columbia
Year: 2009

description: The 70 kDa ribosomal protein S6 kinase (p70s6k) has been implicated in the control of cell proliferation and the onset of protein synthesis following mitogenic and hormonal stimulation. Due to the importance of this role in signal transduction we undertook to purify and characterize the echinoderm isoform of p70s6k, and to evaluate the role of p70s6k in a mammalian system. A rapid protocol was developed for the enrichment of a catalytically active isoform (p52S6K) of the S6 kinase from oocytes of the sea star Pisaster ochraceus. The enzyme was purified ~50,000 fold from oocyte cytosol with a specific enzyme activity of 1.6 µmol per min per mg. The enriched preparation was characterized with respect to ion dependence, phosphotransferase activity and consensus sequence for substrate phosphorylation, determined to be RXXSXR, which was partially distinct from that reported for mammalian p70s6k. Phosphoamino acid analysis of the 40S ribosomal protein S6 phosphorylated by partially purified p52S6K yielded incorporation of ³²P exclusively on serine residues, while 2-dimensional phosphopeptide analysis revealed phosphorylation on at least 8 distinct tryptic peptides, P70S6K consistent with previous studies of mammalian p70S6K. Inhibition of sea star p52S6K phosphotransferase activity after treatment with protein-serine/threonine phosphatases confirmed that p52S6K was regulated by phosphorylation. The p52S6K co-purified with the regulatory and catalytic subunits of protein phosphatase 2A and the heat shock protein 60. A variety of cellular stresses are known to affect the activity of protein kinases. It was demonstrated that protein levels of sea star S6 kinase decreased in response to the stress of heat shock, in a time-dependent manner. In collaboration with Dr. Jing-Song Tao (Ph.D. candidate), I went on to evaluate how p70S6K was affected in mammalian Pam212 keratinocytes in which stress pathways were activated by treatment with photodynamic therapy (PDT) with benzoporphyrin derivative mono-acid ring A (BPD-MA). We phosphotransferase activity of p70S5K in cells maintained in serum-containing medium. In serum-starved Pam212 cells, PDT prevented the insulin-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase), protein kinase B (PKB) and p70S6K. PDT supressed the activation of p70S 6K induced in response to anisomycin and UV irradiation. The activation of p70S6K by these stimuli was found to be independent of PI 3-kinase and PKB. Therefore, inhibition of p70S6K may represent a key signaling event mediating PDT-induced apoptosis. The key novel findings of the research presented here include a substantial enrichment of an echinoderm homologue of p70S6K, and analysis of the regulatory properties of the kinase. We also show, for the first time, that p70S6K is profoundly inhibited in response to PDT, although we do not at present understand the mechanism of PDT-induced signaling.