Annexin II tetramer (AIIt) is a Ca(2+)-dependent phospholipid-binding phosphoprotein. In cells either expressing transforming protein tyrosine kinases or treated with growth factors such as PDGF, AIIt has been shown to contain increased levels of phosphotyrosine. Therefore, we have examined the effects of the in vitro phosphorylation of AIIt by pp60c-src on several activities of the protein. AIIt was phosphorylated by pp60c-src to 0.91 +/- 0.07 mol of phosphate/mol of AIIt (mean +/- SD). The protein tyrosine phosphorylation of AIIt completely inhibited the ability of the protein to bind to and bundle F-actin. In contrast, the phosphoprotein and native protein bound to purified adrenal medulla chromaffin granules with similar affinity; however, the chromaffin granule bridging activity of the phosphoprotein was abolished. The inhibition of the chromaffin granule bridging activity of the phosphoprotein could be partially reversed by the addition of millimolar Ca2+. Furthermore, the phosphorylation of AIIt by pp60c-src inhibited the in vitro ability of this annexin to form a complex consisting of plasma membrane, chromaffin granules, and AIIt. In addition to binding to biological membranes, some annexin proteins have been shown to possess carbohydrate-binding activity. Although native AIIt bound to a heparin affinity column, tyrosine phosphorylation of AIIt blocked the ability of the protein to bind to the heparin affinity column. These results suggest that the tyrosine phosphorylation of AIIt is a negative modulator of AIIt and that the dephosphorylation of AIIt might be necessary for activation of the protein.

Annexin I is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins. The ability of this protein to aggregate and to mediate the fusion of various types of vesicles has supported the hypothesis that this protein might be involved in intracellular membrane fusion processes such as exocytosis. Although annexin I has been described as a major in vitro substrate of both protein kinase C and the epidermal-growth-factor-receptor protein tyrosine kinase, the functional consequences of these phosphorylation events have not been investigated. In this paper we examine the effect of the phosphorylation of annexin I by protein kinase C on the phospholipid aggregation activity of the protein. The stoichiometry of phosphorylation of the protein was affected by the method of preparation of the phospholipid. Under optimal assay conditions protein kinase C catalysed the incorporation of 2.83 +/- 0.23 mol of phosphate/mol of annexin I (mean +/- S.E.M., n = 21). Studies with the Ca(2+)- and phospholipid-independent form of protein kinase C suggested that the phosphorylation of annexin I was stimulated by phospholipid in the absence of Ca2+, although maximal phosphorylation was achieved in the presence of both phospholipid and Ca2+. Phosphorylation of annexin I resulted in a dramatic decrease in the rate and extent of phospholipid vesicle aggregation, without significantly disrupting the binding of the protein to the phospholipid vesicles. The phosphorylation of annexin I increased the EC50 (Ca2+) of phospholipid vesicle aggregation from 19 +/- 10 microM (mean +/- S.D., n = 7) for the native protein to 290 +/- 95 microM (mean +/- S.D., n = 5) for the phosphorylated protein. These results suggest that protein kinase C may act to inhibit the phospholipid vesicle aggregation activity of annexin I.

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(AIIt) 1 is an abundant annexin which is composed of two M r 36,000 annexin II subunits and two M r 11,000 subunits. The 36-kDa subunit consists of two functional domains. The first, the amino-terminal regulatory domain contains the first 30 amino acids of the amino terminus of the heavy chain, incorporates the serine and tyrosine-phosphorylation sites and the binding site for the p11 light chain. The remaining carboxyl domain, comprises the binding sites for Ca 2ϩ

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