Existence of phosphorylated and dephosphorylated forms of cytosolic thymidine kinase (TK1)

In this study we examine whether different TK1 variants of pI 6.9 and 8.3 found by isoelectric focusing gel electrophoresis (IFE) reflect just a phenotype difference due to phosphorylation modifications or have a real phenotypic background. The phosphorylation degree of purified TK1 variants was analyzed by determining the changes in the pI values after treatment with alkaline phosphatase, using IFE. The genetic origin of the two TK1 variants was studied by determining their mol wt. by means of SDS-gelelectrophoresis. Furthermore, the subcellular distribution of the two TK1 variants was also studied. Alkaline phosphatase treatment changed the pI value of purified TK1 from 6.9 to 8.3. No change in the pI value was found when purified TK1 corresponding to pI 8.3 was treated in the same way. Similar results were obtained when treated a cytosolic fraction with alkaline phosphatase. Antibody raised against the C-terminal part of human TK1 only recognized the dephosphorylated TK1 variant corresponding to pI 8.3. There was no difference in the molecular weight between the two TK1 variants. Thus, we concluded that the TK1 variants corresponding to pI 6.9 and 8.3 are of the same genetic origin, but consist of phosphorylated and dephosphorylated forms.