Identification of an AHNAK binding motif specific for the Annexin2/S100A10 tetramer.
The Annexin2 tetramer (A2t), which consists of two Annexin2 molecules bound to a S100A10 dimer, is implicated in membrane-trafficking events. Here, we showed using a yeast triple-hybrid experiment and in vitro binding assay that Annexin2 is required for strong binding of S100A10 to the C-terminal domain of the protein Ahnak. We also revealed that this effect involves only the Annexin2 N-terminal tail, which is implicated in S100A10/Annexin2 tetramerization. The minimal A2t binding motif (A2tBP1) in Ahnak was mapped to a 20-amino acid peptide, and this peptide is highly specific for A2t. We also identified a second A2t binding motif (A2tBP2) present in the N-terminal domain of Ahnak, which binds to A2t, albeit with less affinity. When overexpressed as an EGFP fusion protein in MDCK cells, A2tBPs cofractionate in a calcium-dependent manner and co-immunoprecipitate with S100A10 and Annexin2. In living cells, A2tBPs target EGFP to the cytoplasm as does Annexin2. In response to oxidative and mechanical stress, EGFP-A2tBPs relocalize within minutes to the plasma membrane; a behavior shared with Annexin2-GFP. These results suggest that the A2t complex exists within the cytoplasm of resting living cells and that its localization at the plasma membrane relies on cellular signaling. Together, our data demonstrate that A2tBP1 is a specific A2t complex binding domain and may be a powerful tool to help elucidate A2t structure and cellular functions.
De Seranno
Sandrine
S
INSERM, EMI01-04, Grenoble, France.
Benaud
Christelle
C
Assard
Nicole
N
Khediri
Sami
S
Gerke
Volker
V
Baudier
Jacques
J
Delphin
Christian
C
D020816:Amino Acid Motifs; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D009418:S100 Proteins / Q000737:chemistry / Q000378:metabolism*; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D049452:Green Fluorescent Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10; C075176:AHNAK protein, human; C485184:enhanced green fluorescent protein