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

Abstract

Understanding the regulatory interactions among the 16 subunits of the (αβγδ)4 phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells α, β, γ, and δ subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding αγδ trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and αγδ exhibited only a very low Ca2+-independent activity and a Ca2+-dependent activity similar to that of the native holoenzyme with [Ca2+]0.5 of 0.4 μM for the RM PhK, 0.7 μM for the rPhK, and 1.5 μM for the αγδ trimer. The RM PhK, rPhK, and αγδ subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the α–γ intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant αγδ subcomplex. Our results indicate that both the rPhK and αγδ subcomplex are promising models for future structure–function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.

Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (αβγδ)4 Phosphorylase Kinase Complex

“… a Stable αγδ subcomplexes have been isolated after expression in insect cells (58) and after dissociation of the (αβγδ) 4 complex with LiBr (48). …”

“…For instance, binding of Ca 2+ to δ causes a mass redistribution on the (αβγδ) 2 lobe tips (4) where the carboxy-terminal region of α has been localized (9), and it also alters proteolysis of α near its carboxy-terminus (32). Moreover, the catalytic activities of (αβγδ) 4 and αγδ are stimulated in parallel by Ca 2+ (49, 58). There is strong indirect evidence that the effects on α of the binding of Ca 2+ to δ are mediated by γ, specifically by the γCRD, and adjacent regions of its primary structure have been shown to interact with α and δ within PhK.…”

Urinary Signatures of Renal Cell Carcinoma Investigated by Peptidomic Approaches

“…Many GPCRs are over-expressed in various cancer types and they are constitutively active in malignant cells causing an aberrant response to various signals [63] . The protein Phosphorylase b kinase regulatory subunit α is a key regulatory enzyme of glycogen metabolism [64] . Glycogen can be broken down rapidly when glucose is needed, and Phosphorylase b kinase switches on another enzyme called glycogen phosphorylase b by converting it into the more active form, glycogen phosphorylase a. Alteration of KPB1 seems to be associated with muscle phosphorylase b kinase (PHK) deficiency, a rare disorder caused by mutations in the gene coding for this protein [65] .…”

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

“…Within the (␣␤␥␦) 4 PhK complex, the structure of ␤ is undoubtedly influenced by subunit interactions with itself and with the other three subunits, as evidenced by chemical cross-linking (7,29,30,38,54), EM three-dimensional reconstructions (10), and partial proteolysis (26). As opposed to the ␣, ␥, and ␦ subunits, which have been isolated successfully either individually (8,55,56) or in subcomplexes of PhK (57,58), the ␤ subunit appears to be stable or isolable only in the context of the intact (␣␤␥␦) 4 complex. Although the ␤ subunit undoubtedly influences the structure of ␣, ␥, and ␦ in the PhK complex, we have shown that features of an established Ca 2ϩ -dependent communication network among the ␣, ␥, and ␦ subunits are the same in both the (␣␤␥␦) 4 and ␣␥␦ complexes (57), suggesting a similar structural architecture for these subunits in each complex.…”