Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor.
The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.
Rosorius
O
O
Institute of Biochemistry II, University of Göttingen, Germany.
Mieskes
G
G
Issinger
O G
OG
Körner
C
C
Schmidt
B
B
von Figura
K
K
Braulke
T
T
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D003600:Cytosol / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D006801:Humans; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D010957:Plasmids; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D004274:DNA, Recombinant; D009842:Oligopeptides; D010446:Peptide Fragments; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D011493:Protein Kinase C