The γ Subunit of Phosphorylase Kinase Contains a Pseudosubstrate Sequence

The catalytic subunit, γ, of phosphorylase kinase is regulated by a complex set of interactions involving the calcium‐binding protein calmodulin and two other subunits designated α and β. These interactions regulate γ activity that, at least for the calmodulin interactions, involves the regulatory domain in γ spanning residues 302–366. Within this regulatory domain, we report the identification of a sequence (residues 326–334) that resembles the phosphorylation site in γ substrates with the exception that a V residue (V332) occurs at the analogous position of the phosphorylated S/T residue. The inhibitory properties of the sequence were assayed with a 10‐amino‐acid peptide of the sequence. This peptide inhibits a truncated version of γ, residues 1–300, which is missing the regulatory domain, more potently than it inhibits full‐length γ, and it is a better inhibitor of the full‐length γ at pH 8.2 than at pH 6.8. A similar peptide of the same sequence, except for a S substitution of the V residue, is a good substrate with a comparable K m and better V max than peptides of similar length that represent the phosphorylation site in the substrate of the enzyme, glycogen phosphorylase. A mutant γ protein, with a S for V332 substitution ([V332S]γ), was prepared using the baculovirus expression system. [V332S]γ autophosphorylates by an intramolecular mechanism. This demonstrates that this sequence can occupy the catalytic site in the protein. Development of [V332S]γ affords an experimental model in which the effects of the regulatory factors on autophosphorylation can be determined.