| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 97 === Interleukin-6 (IL-6) can induce many signal transduction pathways in multiple myeloma cell (MM cell), and the phosphatidylinositol 3-kinase / Akt kinase pathway (PI3K / AKT pathway) can promote proliferation of multiple myeloma cells and inhibit cell apoptosis. Activated AKT plays the most important role in those reactions. In previous 2-D gel electrophoresis analysis, we suspected that the position shift of β-actin is due to its posttranslational modification by AKT. Therefore, the first object is to confirm this hypothesis by more 2-D gel electrophoresis & MALDI-TOF. AKT specifically phosphorylates its substrates at Ser or Thr residue of the RXRXX(S/T) motif and Phospho-(Ser/Thr) Akt substrate antibody (PAS antibody) can recognize the RXRXXp(S/T) peptide motif. Taking advantage of that characteristics, we used PAS antibody to detect Akt substrate. We found that beta-actin was one of the proteins recognized by PAS antibody. We then used PAS antibody and β-actin antibody, reciprocally, in immune precipitation & Western blot analysis to confirm that β-actin is a substrate of AKT. The monomer actin, beta-actin, will polymerize to form the filamentous-actin (F-actin) under the right conditions. Actin plays an important role in cytoskeletal organization & cell motility of cells. To study the significance of the interaction between β-actin and AKT in AF-10, we used flow cytometer to measure the amount of F-actin labeled with phalloidin-FITC after inducing AF-10 with IL-6. We found that IL-6 enhanced the F-actin formation and wortmannin inhibited it. We also showed that IL-6 induced the migration of AF-10. Interestingly, after treating AF-10 with wortmannin, we found the cellular migration ratio of this group is higher than that of the group treated with IL-6 and of the control group。In summary, we found that in AF-10 cells, the interaction between β-actin and AKT indeed is influenced by PI3K / AKT pathway which is induced by IL-6. And the amount of F-actin is increased with IL-6 treatment. But we need further studies to illuminate the mechanisms of IL-6-induced cell migration of myeloma cells.
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