Role of the amino-terminal domain in regulating interactions of annexin I with membranes: Effects of amino-terminal truncation and mutagenesis of the phosphorylation sites

Abstract

Phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of bovine or human annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold (Wang, W., & Creutz, C. E. (1992) Biochemistry 31, 9934-9936). In the present study three forms of human annexin I truncated in the amino terminus at residue Trp-12, Lys-26, or Lys-29 exhibit dramatic differences in their sensitivities to calcium in a chromaffin granule aggregation assay, while the [Ca2+](1/2)max values for binding of the truncated proteins to granule membranes are similar. Cleavage at Trp-12 causes a 3-fold decrease in calcium sensitivity in the membrane aggregation assay, while cleavage at Lys-26 causes a 4-fold enhancement of calcium sensitivity. In contrast, cleavage at Lys-29 results in virtually no change in calcium sensitivity. Mutagenic substitution with negatively charged amino acids of Ser-27, a site for phosphorylation by protein kinase C, or Tyr-21, a site for phosphorylation by the epidermal growth factor receptor kinase, mimics the inhibition of granule-aggregating activity seen with phosphorylation by protein kinase C. When bovine chromaffin cells are stimulated to secrete by nicotine, annexin I is phosphorylated in the amino terminus. Thr-24 and Ser-28, which are sites for phosphorylation by protein kinase C in vitro, are two of the sites phosphorylated in vivo in stimulated chromaffin cells. These data demonstrate that the ability of annexin I to promote membrane aggregation is highly sensitive to changes in the structure of the N-terminal domain of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Two Proteins Modulating Transendothelial Migration of Leukocytes Recognize Novel Carboxylated Glycans on Endothelial Cells

“…For example, it has been shown by several studies that the N-terminal peptide Ac 2-26 mimics the potent antiinflammatory effects of intact annexin I (35). The amino-terminal domain regulates interactions with membranes (37), and is directly involved in binding to S100 proteins (38,39). Most notably, the N-terminal peptide Ac2-12, which in our study appears essential for binding to the novel glycans (Fig.…”

Requirement for Annexin A1 in Plasma Membrane Repair

“…One is the "N-terminal interaction domain," an N-terminal sequence of ϳ40 residues that is unique in annexin A1 and binds several proteins including S100A11, a member of the S100 family (12). Peptides of the N-terminal interaction domain have previously been shown to disrupt interactions of annexins with specific protein ligands, and perhaps most importantly for the present study, the N-terminal domain is required for annexin A1 membrane aggregation activity (20). Therefore, we next employed as a potential function-inhibiting reagent a peptide comprising the N-terminal 1-13 residues of annexin A1.…”

A Proinflammatory Role for Proteolytically Cleaved Annexin A1 in Neutrophil Transendothelial Migration

“…These data indicate that calpain I proteolysis of AnxA1 was essential for generating an active AnxA1 product that could activate ERK in HUVEC, and the molecular mass of the active form of ∼33 kDa suggests that the C-terminal fragment is the active peptide. To affirm this finding, we also tested the N-terminal AnxA1 peptide, Ac2-26, that would be generated by calpain 1 cleavage (15) and has been shown previously to mimic the known antiinflammatory functions of the full-length protein (16,17). We found that it was not able to activate endothelial ERK (Fig.…”

“…Therefore, the N terminus of the active truncated AnxA1 could be between aa 27 and 30. AnxA1 has been shown previously to be cleaved at various sites in the N terminus by a number of proteases, including calpain I and plasmin after Lys-26 and Lys-29 (15), respectively. To test whether the active truncated AnxA1 could be generated by calpain cleavage, fulllength recombinant AnxA1 was treated with human calpain I, and the resulting product was assayed for its ability to activate ERK in HUVEC.…”