ANNEXIN-5 AS A POTENTIAL REGULATOR OF ANNEXIN-1 PHOSPHORYLATION BY PROTEIN-KINASE-C - IN-VITRO INHIBITION COMPARED WITH QUANTITATIVE DATA ON ANNEXIN DISTRIBUTION IN HUMAN ENDOTHELIAL-CELLS

In vitro phosphorylation of annexin 1 by purified rat brain protein ki nase C (PKC) has been studied in the presence of annexin 5, which is n ot a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing t he concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annex in 5 and the residual phosphorylation of annexin 1. These data fit wit h the 'surface depletion model' explaining the anti-phospholipase acti vity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have be en quantified in human endothelial cells by measuring the radioactivit y bound to the proteins after Western blotting with specific antibodie s and I-125-labelled secondary antibody. Our data indicate that annexi ns 1 and 5, PKC and PtdSer are present in human endothelial cells in r elative amounts very similar to those used in vitro under conditions p ermitting the detection of the inhibitory effect of annexin 5. Since a nnexin 1 remained refractory to PKC-dependent phosphorylation in intac t cells, we suggest that annexin 5 might exert its inhibitory effect t owards PKC in vivo, provided that its binding to phospholipids can occ ur at physiological (micromolar) concentrations of Ca2+. This was prev iously shown to occur in vitro using phosphatidylethanolamine/phosphat idic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-20 0]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation wit hout interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PK C. Whether a similar mechanism also occurs in vivo remains to be deter mined.