To study the role of protein kinase Cα in cell growth and differentiation using the TAT-mediated protein transduction system

• Main Authors: Ya-Huei Shiau, 蕭雅蕙
• Other Authors: Wey-Jinq Lin
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/32019013085819833274

碩士 === 國立陽明大學 === 生物藥學研究所 === 95 === Protein kinase C is a family of serine/threonine kinases implicated in modulation of various cellular processes cell proliferation, differentiation, apoptosis. However, the functional roles of each isozyme of the family have not been conclusively elucidated due to the high similarity in regulatory and biochemical properties. Protein transduction domains have been shown to be highly efficient in delivering proteins into cells in a non-invasive manner. This system facilitates exploring gene functions especially for those that are difficult to transfect. In this study, I first expressed the recombinant TAT-fused protein kinase Cα in E. coli. The majority of the recombinant protein was in inclusion bodies. A denaturation/renaturation protocol was successfully developed to obtain soluble TAT-PKCαa proteins. The recombinant TAT-PKCαa protein entered human multi-potent leukemia cell line K562、human fetal osteoblast progenitor cells (hFOB)、human mesenchymal stem cells (MSCs) in a time- and dose-dependent fashion as shown by Western blot analysis. Introduction of TAT-PKCαa significantly enhanced megakaryocytic differentiation of K562 cells in a kinase activity-dependent manner. However, the effect was suppressed by application of conventional PKCα inhibitor Gö6976 or MAPK signal pathway inhibitor PD98059. Mesenchymal stem cells (MSCs) readily differentiate to multiple connective tissue lineages, including osteoblast, chondrocytes, and adipocytes by different stimulations, and it are of great application potentials in cell therapy, tissue engineering and regenerative medicine. Understanding, in depth, of the molecular events controlling MSC proliferation and differentiation will definitely facilitate the development of a more appropriate strategy. The TAT-PKCa protein was able to enter mesenchymal stem cells (MSCs) efficiently. Moreover, TAT-PKCα enhanced osteogenic differentiation of MSC as measured by the alkaline phosphatase (ALP) activity, as early maker of osteo-differentiation after osteo-differentiation medium treatment.