Abstract
In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with (32)P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, 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 modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.
Highlights
In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase
Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with 32P, and the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis
Proteins of CHO cells transfected with the pSecFAPPw vector were metabolically labeled with [32P]orthophosphoric acid
Summary
Intracellular traffic from the endoplasmic reticulum to the trans-Golgi network, BSDL is associated with intracellular membranes [9, 10], from which the enzyme is dissociated upon the phosphorylation of a hydroxylated amino acid residue of the protein, such as threonine or serine, by the action of a protein kinase casein kinase II [11] This third post-translational modification remains poorly documented, but it appeared essential to the secretion of the enzyme [12] and should occur once the sequential glycosylation of the protein was achieved. We used a vector including the cDNA encoding FAPP that leads to a secreted protein upon transfection in CHO cells [19]
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