The γ-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin

Abstract

Phosphorylase kinase is a Ca²⁺-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the δ-subunit). Ca²⁺-dependent activity of the enzyme is thought to be regulated by direct interaction of the δ-subunit with the catalytic subunit (the γ-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (δ-subunit)-binding domain(s) in the γ-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the γ-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca²⁺/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the γ-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured γ-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca²⁺-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the γ-subunit and calmodulin (δ-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca²⁺ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the γ-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.