|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1997 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
The object of this study is to make clear the activation mechanism and physiological function of non-receptor type protein-tyrosine kinase p72^<syk> in the proliferation of hematopoetic cells. I investigated the activation and regulation mechanism of of p72^<syk> by insulin and IGF-1 stimulation in lymphoblast cell line IM-9 overexpressing insulin receptors. It was revealed that p72^<syk> exists in IM-9 cells, and the activity of auto-phosphorylation of tyrosine residue of p72^<syk> increased within 15-30 sec by insulin or IGF-1 stimulation in a dose-dependent manner, and then gradually reduced within 2-5 min. To examine whether p72^<syk> is associated with insulin receptor, I performed co-immunoprecipitation assay. Using western blotting insulin recepter was slightly detected in co-immunoprecipitates with anti-syk antibody, while p72^<syk> was not in in co-immunoprecipitates with anti-insulin recepter antibody.
Next, we examined the protein-tyrosine phosphorylation of p72^<syk> and i
nsulin receptor by the stimulation of H_2O_2, which has the mimic effect of insulin and is inhibitor of protein-tyrosine phosphatase. The phosphotyrosine of insurin receptor and p72^<syk> was remarkably increased by H_2O_2 within 1-3 min. And the anti-phosphotyrosine immuno-blots of anti-insulin recepter immunoprecipitates showed tyrosine phospho-rylation of 95kDa and 72-74 kDa proteins. We investigated the effect of cAMP-elevating agents, dibutyryl cAMP and forskolin on protein-tyrosine phosphorylation of p72^<syk> by H_2O_2 stimulation. H_2O_2-induced protein-tyrosine phosphorylation and autophosphorylation of p72^<syk> were suppressed by pre-treatment with dibutyryl cAMP,but on the contrary the protein-tyrosine phosphorylation of p72^<syk> increased by pre-treatment with forskolin. We are investigating the mechanism of the different effects between dibutyryl cAMP and forskolin on the tyrosine-phosphorylation of p72^<syk> in H_2O_2-stimulated IM-9cells.
As a part of this study, we cooperatively studied the effect of interleuukin-3 (IL-3) stimulation for the activation of p72^<syk> in myeloid leukemia cell line, AML193, which proliferates depending on IL-3 or granulocyte colony stimulating factor (G-CSF).
Some human leukemia cell lines, especially AML 193, contained a large amount of p72^<syk>, which was immediately activated by the stimulation with IL-3 or G-CSF.We futher investigated the relation of p72^<syk> and IL-3 with IL-3-mediated signaling. The IL-3 receptor beta subunit was co-immunoprecipitated with p72^<syk>. Since the IL-3 receptor beta subunit is known to mediate growth signal, our results indicate that p72^<syk> may be involved in the proliferation of myeloid cells. Less