Phosphorylation of the oncofetal variant of the human bile salt-dependent lipase - Identification of phosphorylation site and relation with secretionprocess

In this paper, we report, for the first time, the localization of the phosp horylation site of the fetoacinar pancreatic protein (FAPP), which is an on cofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radi olabeled the enzyme with P-32, and then the protein was purified by affinit y chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatograp hy, associated with the radioactivity profile, revealed that the phosphoryl ation site is located at threonine 340, Site-specific mutagenesis experimen ts, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the m odification on the activity and secretion of the enzyme. These studies show ed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of t heir ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartmen t, However, the mutated enzyme, which was sequestrated in the transfected C HO cells, remains active on bile salt-dependent lipase substrates.