Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3.
A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.
Ferrarese
Anna
A
Venetian Institute of Molecular Medicine, via Orus 2, 35129 Padova, Italy.
Marin
Oriano
O
Bustos
Victor H
VH
Venerando
Andrea
A
Antonelli
Marcelo
M
Allende
Jorge E
JE
Pinna
Lorenzo A
LA
D025601:Adenomatous Polyposis Coli Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D020035:Holoenzymes / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D014170:Transformation, Genetic; D051176:beta Catenin / Q000378:metabolism
D025601:Adenomatous Polyposis Coli Protein; D020035:Holoenzymes; D007527:Isoenzymes; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D051176:beta Catenin; D012694:Serine; D001120:Arginine; D047389:Casein Kinase I; D038362:Glycogen Synthase Kinase 3; D003545:Cysteine