ACTIVATED PROTEIN-KINASE-C-ALPHA ASSOCIATES WITH ANNEXIN-VI FROM SKELETAL-MUSCLE

We have previously detected a number of protein kinase C (PKC) alpha-b inding proteins in skeletal muscle cytosol by blot overlay assay, and now identify the major, 69 kDa binding protein as annexin VI by immuno blotting and overlay assay of hydroxyapatite chromatography fractions. Annexin VI was also detected in immunoprecipitates of PKC alpha. Anne xin VI and PKC alpha are both calcium-dependent phospholipid-binding p roteins, and detection of the interaction was dependent on the presenc e of calcium and phosphatidylserine (PS). The association probably inv olves specific protein-protein interactions rather than mere bridging by lipid molecules: firstly, detection of PKC alpha-annexin VI complex es by overlay assay was not diminished when PS concentrations were inc reased over a 10-fold range, while that of other PKC alpha-binding pro tein complexes was reduced or abolished; secondly, the presence in the overlay assay of a PKC pseudosubstrate peptide, analogous to a PKC se quence previously found to be involved in PKC binding activity, reduce d complex formation; thirdly, we were also able to detect annexin VI i nteraction with PKC beta by overlay of skeletal muscle cytosol, but no t with PKC theta, the major novel PKC in this tissue, suggesting seque nces specific to calcium-dependent PKC isoenzymes are involved. While other annexin isoforms may be PKC substrates or inhibitors, annexin VI phosphorylation by PKC alpha could not be detected after co-purificat ion, while phosphorylation of subsequently-added histone IIIS was read ily observed. Annexin VI is a major skeletal muscle protein and our da ta are consistent with a role for this isoform in the control of calci um-dependent PKC.