1997 Fiscal Year Final Research Report Summary
Signal transduction mechanism through non-receptor type protein-tyrosine kinase, p72^<syk> in proliferation of hematopoietic calls
| Project/Area Number |
07680701
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Functional biochemistry
|
|---|
| Research Institution | Fukui Prefectural University College of Nursing |
|---|
Principal Investigator |
ASAHI Momoyo Fukui Prefectural University College of Nursing, Assistant professor, 助教授 (60100624)
|
|---|
| Project Period (FY) |
1995 – 1997
|
| Keywords | Insulin Receptor / IGF-1 Receptor / Protein Tyrosine Kinase p72^<syk> / Dibutyryl cAMP / Forskolin / H_2O_2 / IL-3 / IL-3 recepter beta subunit |
|---|
| Research Abstract |
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 ins
… More
ulin 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
|
