The tight association of the tyrosine kinase substrate annexin II with the submembranous cytoskeleton depends on intact p11- and Ca(2+)-binding sites.
Annexin II, a member of the annexin family of Ca(2+)- and lipid-binding proteins, is a major substrate of the pp60src kinase. It is unique within the annexin protein family, since it can form a tight heterotetrameric complex with the cellular protein ligand p11, a member of the S100 protein family. Within the cell, the annexin II2p11(2) complex is localized at the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton. This intracellular localization is thought to be the consequence of a typical annexin II property observed in vitro, its Ca(2+)-dependent binding to phospholipids and cytoskeletal elements (F-actin, non-erythroid spectrin). We employed site-directed mutagenesis to create mutant annexin II molecules with defects either in the p11-binding site or in the Ca(2+)-binding sites present in repeats 2, 3 and 4. The mutated annexin II derivatives were expressed in HeLa and RMCD cells by transfection of the appropriate DNA constructs in order to analyze the importance of p11- and Ca(2+)-binding for the intracellular localization of annexin II. Immunofluorescence microscopy with a monoclonal antibody that specifically detected the transfected annexin II derivatives indicated that the Ca(2+)-dependent incorporation of annexin II into the submembranous network depended on its ability to form the annexin II/p11 complex and on the presence of intact Ca(2+)-binding sites. Neither monomeric annexin II lacking an intact p11-binding site, nor the annexin II mutant with defects in the Ca(2+)-binding sites in repeats 2, 3 and 4 were associated with the Triton X-100-resistant network of the submembranous cytoskeleton.
Thiel
C
C
Max Planck Institute for Biophysical Chemistry, Goettingen, FRG.
Osborn
M
M
Gerke
V
V
D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D009418:S100 Proteins; D014162:Transfection; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D002118:Calcium; D010455:Peptides; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10