INHIBITORY EFFECT OF ANNEXIN-V ON PROTEIN-KINASE-C ACTIVITY IN MESANGIAL CELL LYSATES

Annexin V belongs to a large family of calcium-binding and phospholipi d-binding proteins and may act as an endogenous regulator of the prote in kinase C (PKC) activity. This study examines the effect of annexin V on the in vitro PKC activity in cultured mesangial cells using histo ne H1, the peptide [Ser25]PKC-(19-31), or endogenous proteins as subst rates. The SDS/PAGE pattern of P-32-labeled mesangrial proteins showed that the calcium-independent PKC [(n+a)PKC] phosphorylated several pr oteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional pr oteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium-dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylat ion of (n+a)PKC substrates. By contrast, specific phosphorylation of p roteins and annexin I by cPKC, was reduced in a dose-dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibit ed the phosphorylation of histone H1 or peptide [Ser25]PKC-(19-31) by cPKC. Moreover, removal of annexin V from cytosols increased the annex in I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal-transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.