Activation and inhibition of phosphorylase kinase by monospecific antibodies raised against peptides from the regulatory domain of the γ-subunit

Abstract

The C terminus of the catalytic γ-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346) and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the γ•δ complex. Antibodies raised against the very C terminus of the γ-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the γ•δ complex, in the presence and absence of Ca²⁺. In the presence of Ca²⁺ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the γ•δ complex 2.5-fold. Activation of the holoenzyme and the γ•δ complex by anti-PhK5 was 50-70 % of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the γ•δ complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the γ-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity.