Phosphorylation of the oncofetal variant of the human bile salt-dependent lipase. identification of phosphorylation site and relation with secretion process.
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.
Verine
A
A
INSERM Unité 260, Faculté de Médecine, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 5, France.
Le Petit-Thevenin
J
J
Panicot-Dubois
L
L
Valette
A
A
Lombardo
D
D
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008049:Lipase; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D002787:Sterol Esterase / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D006023:Glycoproteins; D013912:Threonine; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; C054621:CEL protein, human; D008049:Lipase