Unphosphorylated alpha-PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro.

Expression of the alpha-isoform of protein kinase C (alpha-PKC) in E. coli yielded the unphosphorylated 74 kD precursor molecule. This precursor form exhibited phospholipid- and calcium-dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity. In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of alpha-PKC is not required for binding of substrates, cofactors, or activators. These results, therefore, suggest that posttranslational phosphorylation of alpha-PKC is not needed for the formation of a functional enzyme-substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.