Antibodies to the N-terminus of calpactin II (p35) affect Ca2+ binding and phosphorylation by the epidermal growth factor receptor in vitro.
Calpactins I and II are related 39-kilodalton (kDa) proteins that interact with phospholipids and actin in a calcium-dependent manner and are substrates of tyrosine protein kinases. They contain a short amino-terminal tail attached to a 36-kDa core domain. Monoclonal antibodies (Mabs) were raised to bovine calpactin II and used as site-specific probes of its structure and function. All of the antibodies reacted with native calpactin II and gave rise to a single band of 39 kDa among total cell protein displayed on Western blots. Most of the antibodies (9/14) reacted with determinants on the tail as shown by Western blots and competition with a synthetic tail peptide. Four antibodies reacted with determinants on the core and a 10-kDa tryptic fragment. Antibody-calpactin II complexes were tested for their ability to interact with lipid, actin, and Ca2+ and to serve as substrates of the epidermal growth factor (EGF) receptor tyrosine protein kinase. Whereas none of the antibodies had a detectable effect on actin binding, two anticore antibodies reduced calpactin's affinity for phospholipid. Ca2+-binding sites are known to reside within the core region, yet most antitail antibodies markedly increased the affinity of calpactin II for Ca2+, with four Ca2+-binding sites observed. Antitail antibodies either (i) abolished or (ii) greatly stimulated (10-fold) the phosphorylation of calpactin II by the EGF receptor. These results suggest that the interactions between calpactin II and Ca2+, phospholipid, or the EGF receptor are more complex than previously thought and can be modulated by interactions occurring in the tail.
Glenney
J
J
Molecular Biology and Virology Laboratory, Salk Institute for Biological Studies, San Diego, California 92138.
Zokas
L
L
D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism
D017302:Annexins; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002135:Calcium-Binding Proteins; D000939:Epitopes; D010446:Peptide Fragments; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D002118:Calcium