The functional roles of cyclin-dependent kinase 5 (Cdk5) activators in neuronal and muscle cell differentiation
by Kun Lam
THESIS
1999
Ph.D. Biochemistry
xiii, 168 leaves : ill. (some col.) ; 30 cm
Abstract
RT-PCR analysis of cultured non-neuronal cell lines indicated absence of the Cdk5 activator gene expression. Similar analysis of cultured neuronal cell lines revealed low or no expression of p35nck5a mRNA and moderate p39nck5ai mRNA expression in PC-12 cells. On the other hand, cultured neuronal cells that were induced to differentiate contained p35nck5a mRNA and increased p39nck5ai mRNA expression in two of the three cell lines examined. In addition, proteins extracted from NG 10% 15 differentiated cells had kinase activity profile comparable to rat brain. nck5anck5ai...[ Read more ]
RT-PCR analysis of cultured non-neuronal cell lines indicated absence of the Cdk5 activator gene expression. Similar analysis of cultured neuronal cell lines revealed low or no expression of p35nck5a mRNA and moderate p39nck5ai mRNA expression in PC-12 cells. On the other hand, cultured neuronal cells that were induced to differentiate contained p35nck5a mRNA and increased p39nck5ai mRNA expression in two of the three cell lines examined. In addition, proteins extracted from NG 10% 15 differentiated cells had kinase activity profile comparable to rat brain.
Further experimentation also detected the presence of p35nck5a and p39nck5ai mRNA in rat muscle and in C2C 12 cells and their concentration increased to a maximum between 48 and 60 hr of differentiation. This was accompanied by a concomitant increase in Cdk5 kinase activity. It was also found that while this kinase activity in the proliferating cells was readily demonstrated, it was not attributable to p35nck5a. Hence, other activators may play a role here.
Skeletal muscle formation requires several transcription factors, one of which is Myogenin which binds to a sequence known as E-box in the promoter region of several muscle-specific genes regulating their expression in the earlier stages of differentiation. Gel mobility shift experiments have revealed myogenin to be a phosphorylation substrate of activated Cdk5, which was found to cause reduced E-box binding affinity of myogenin.
Bacterially expressed p35nck5a and p39nck5ai had high kinase activity upon incubation with Cdk5 but lacked activity when associated with Dominant Negative form of Cdk5. Cell transfection experiments showed that Cdk5, p35nck5a,p39nck5ai and myogenin would not on their own induce differentiation but they can enhance morphological differentiation upon serum reduction. In addition, transfection of Cdk5/p35nck5a even in 20% serum resulted in stimulating myotube formation.
Post a Comment