Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

Phosphorylase b kinase (PhK),1 a hexadecameric enzyme complex with (αβγδ)4 subunit stoichiometry (1), controls glycogen degradation via phosphorylation of glycogen phosphorylase b (GPb). The kinase activity, which resides on the catalytic γ subunit, has an obligatory requirement for Ca2+, which binds to PhK’s intrinsic calmodulin (CaM) subunit, δ, and couples muscle contraction with energy production via glycogenolysis (2). In addition to Ca2+, a large number of other activators further enhance PhK’s activity at neutral pH, mostly by binding to allosteric sites on its large, regulatory α and β subunits (2). Phosphorylation of the α and particularly β subunits also markedly stimulates PhK activity at neutral pH (3). Thus the PhK complex has evolved the capacity to have its activity regulated by a diverse group of activators; in fact, summing the masses of its regulatory α (138.4 kDa (4, 5)), β (125.2 kDa (5)), and δ (16.7 kDa (6)) subunits and the C-terminal regulatory domain of its catalytic γ subunit (11.7 of 44.7 kDa (7)), one finds that a full 90% of PhK’s 1.3 MDa mass is noncatalytic, presumably largely devoted to its regulation. The mechanisms through which the diverse allosteric activators acting on the α, β, and δ regulatory subunits stimulate the activity of the catalytic γ subunit of PhK remains for the most part unknown, in large part because structure–function studies using mutagenesis to probe the regulation of the PhK complex have not been feasible. A critical breakthrough in this regard was made 5 years ago with the first reported expression, utilizing baculovirus, of the PhK holoenzyme complex, as well as its αγδ and γδ subcomplexes (8). For practical reasons, that study used separate vectors for each of the four subunits, with α, γ, and δ from rat and β from rabbit. Importantly, the coexpressed subunits of the PhK holoenzyme did assemble in the appropriate subunit stoichiometry and exhibit pH- and Ca2+-dependent activities that were similar to those of PhK purified from fast-twitch skeletal muscle of the rabbit (RM PhK), which is the form of the enzyme for which virtually all reference information has been obtained during the last 55 years (2). There were, however, some key differences between the properties of the expressed recombinant PhK (rPhK) and RM PhK: specific activity at pH 8.2 (25%of that expected); high Ca2+-independent basal activity (50%); Ka for Ca2+ (one-tenth the normal required concentration); no activation upon phosphorylation of the α and β subunits; and pH 6.8/8.2 activity ratio (0.40 instead of<0.05). This last property is significant because the ratio of activities at these two pH values is an accepted indicator of the activation state of wt PhK (9). So, although this first expression system represented a huge step forward and proved the feasibility of producing active, hexadecameric rPhK, the resultant PhK complex was not yet suitable for structure–function studies aimed at understanding the control of PhK activity by its regulatory subunits. In the work herein, we set about improving the previous system, with the goal of producing rPhK having properties that closely mimic those of isolated rabbit skeletal muscle PhK. We focused on three major changes to the published expression system. First, we chose to use cDNA from rabbit for the α, β, and γ subunits. Even though the sequences for the various subunits are highly conserved between rabbit and rat (2), we reasoned that even small differences might affect subunit interactions and regulatory properties. The cDNA used for CaM, the δ subunit, was from Xenopus and codes for CaM with a sequence identical to that of rabbit (10, 11). Second, to allow stoichiometric expression of the four subunits that could be reproducibly maintained, we desired that all four subunits be expressed by a single vector, rather than four as in the published procedure. Third, we decided that when a His6 tag was used, it would be placed on a different subunit than the catalytic γ, as was done previously. We opted for the N-terminus of the α subunit, in that this region has yet to be implicated in any property of PhK. As is described herein, we have effected these three changes and produced a rPhK complex with properties that very closely mimic those of PhK isolated from muscle, thus allowing future structure–function studies on the intersubunit regulation of PhK activity.