Interaction study of b1,4-galactosyltransferase and PITSLRE b1 kinase

In recent years, it was shown that protein bound carbohydrates play crucial roles in many intermolecular and cell-cell interactions. There are a number of proposed mechanisms of the regulation of the glycosylation process including the regulation of the enzymes involved by changing their phosphorylation state. One of the most studied enzymes that transfers carbohydrates to proteins, human b1,4-galactosyltransferase, was already shown to be phosphorylated on serine residue(s). The responsible kinase and how and where the phosphorylation event actually occurs was, though, to date not clearly identified. One of the candidates for phosphorylation of the b1,4-galactosyltransferase is the PITSLRE b1 kinase. This kinase was shown to have regulating role in cell-cycle and programmed cell death and also, apparently, co-purifies with b1,4-galactosyltransferase from milk and is able to alter b1,4-galactosyltransferase activity in vitro and in vivo. It is proposed to study the interaction of the two enzymes and the properties of the kinase more thoroughly. Pure bovine recombinant enzymes will be used to identify to what extent proteins affect each other and whether and where the phoshorylation event actually occurs. Experiments will be performed to identify which part of b1,4-galactosyltransferase interacts with the kinase. Substrate specificity and other key enzymatic properties of the kinase will be studied.