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Cytokine-induced activation of PKC isoenzymes in hemopoietic cell

University of British Columbia

Three cytokines which regulate the differentiation, mitogenesis, and activation of hemopoietic cells, interleukin (IL)-3, IL-5 and granulocyte-macrophage colony stimulating factor (GM-CSF), activate similar signalling pathways. Considerable knowledge regarding JAK-STAT and RAS-RAF pathways exists but, there is little information about the regulation of specific PKC isoforms by each of these cytokines. The first hypothesis was that unique functions of IL-3, IL-5 or GM-CSF result from activation of specific PKC isoforms. It was shown in human erythroleukemia cells, TF-1, that proliferation and tyrosine phosphorylation of similar substrates occurred in response to IL-3, IL-5, or GM-CSF. The results with IL-5 were unique. Growth of cells in IL-5 was necessary for the increased expression of the IL-5 receptor α subunit and responsiveness to IL-5. Another novel finding was that cells grown in IL-5 expressed increased levels of PKCα, δ and ε compared to cells grown in IL-3. Using SDS-PAGE and PKC isoform-specific antibodies, it was found that in cells stimulated with phorbol ester, PKCβ, α and ε translocated to the membrane from the cytosol. However, in response to IL-3, IL-5 or GM-CSF, PKCα and β signal translocation was not apparent. Interestingly, PKCδ and ε underwent band shifts in response to IL-3, IL-5, GM-CSF or phorbol ester. These novel results indicated that activation of PKCδ and ε may not require their translocation to the membrane. The recent findings, that PKCδ, ε and ς could be activated nonspecifically, by lipid products of phosphatidylinositol 3-kinase (PI3-kinase) led to a second hypothesis; that in response to IL-3, IL-5 or GM-CSF, PI3-kinase associates with specific PKC isoforms and somehow regulates their activity. It was found that both PKCδ and ε associated with PI3-kinase in unstimulated TF-1 cells. More importantly, PKCδ (but not PKCε) association with PI3-kinase was increased by GM-CSF treatment of the cells. Results using two inhibitors of PI3-kinase, wortmannin and LY294002, showed that the former inhibited the association while the latter did not, suggesting that PI3-kinase lipid products may not be required for the association. This association of PKCδ with PI3- kinase was also observed in platelets treated with platelet activating factor or thrombin. It was then hypothesized that perhaps the protein kinase activity of PI3-kinase regulates PKCδ activity. This was tested in platelets. Thrombin induced increased PKCδ kinase activity in the PKCδ:PI3-kinase immune complex. Also, within this complex, PKCδ underwent serine phosphorylation in a PI3-kinase protein kinase assay. The PKCδ activity and phosphorylation were inhibited by pre-treatment of platelets with LY294002 and wortmannin. Platelet aggregation by thrombin was also inhibited by LY294002 and wortmannin. In conclusion, these results support a role for an association of PI3- kinase and PKCδ that allows regulation of PKCδ activity by agonists in a novel fashion. Furthermore, this interaction occurs following hemopoietic receptor signalling or serpentine receptor signalling, suggesting one possible convergence of these pathways, at the point of PKCδ regulation.

Thesis/Dissertation

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2009-05-29

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http://hdl.handle.net/2429/8491

Experimental Medicine

University of British Columbia

UBCV

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