Baculovirus-Mediated Overexpression of the Phosphorylase <i>b</i> Kinase Holoenzyme and αγδ and γδ Subcomplexes

Kumar, Priyadarsini; Brushia, Robert J.; Hoye, Elaine; Walsh, Donal A.

doi:10.1021/bi049223i

Cited by 5 publications

(12 citation statements)

References 29 publications

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“…Thus, most of the physicochemical changes observed in this study likely reflect changes in PhK's a and b subunits. Moreover, the changes induced by [pH are most likely triggered by deprotonation of residue(s) on the a and/or b subunits, because the pH 6.8/8.2 activity ratio of the gd dimer approaches unity (Chan and Graves 1982;Kumar et al 2004), whereas that of the (abgd) 4 PhK complex is dramatically lower at ;0.01. Even though the changes induced by Ca 2+ are presumably triggered by PhK's endogenous calmodulin (d) subunit, the affinity of PhK for Ca 2+ is different at pH 6.8 and 8.2 (Brostrom et al 1971;Cohen 1980), and phosphorylation of its a and b subunits also alters PhK's affinity for Ca 2+ (Brostrom et al 1971;Cohen 1980).…”

Section: Discussionmentioning

confidence: 99%

Physicochemical changes in phosphorylase kinase associated with its activation

2008

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Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca 2+ -dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as [pH), but Ca 2+ dependence is retained. Little is known about the structural changes associated with PhK's activation by [pH and Ca 2+ , but activation by both mechanisms is mediated through regulatory subunits of the (abgd) 4 PhK complex. In this study, changes in the structure of PhK induced by [pH and Ca 2+ were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca 2+ and [pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH ;7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 À Ca 2+ ) versus that where it would be most active (pH 8.2 + Ca 2+ ), the joint activation by [pH and Ca 2+ is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of [pH and Ca 2+ in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.Keywords: phosphorylase kinase; Ca 2+ ; Tyr environment; secondary structure; tertiary structure; surface electrostatics; zeta potential Phosphorylase kinase (PhK), a complex regulatory enzyme in the cascade activation of glycogenolysis, is the only known kinase to phosphorylate and activate glycogen phosphorylase. By far, the most is known about the PhK from skeletal muscle (for reviews, see Heilmeyer Jr. 1991; Brushia and Walsh 1999), and that is the subject of this study. This PhK is a 1.3-MDa hexadecameric complex composed of four copies each of a single catalytic protein kinase subunit (g, 44.7 kDa) and three different regulatory subunits (a, 138.4 kDa; b, 125.2 kDa; and d, 17.7 kDa). Thus, its regulatory subunits account for 86% of the mass of the (abgd) 4 PhK complex. The a and b subunits are homologous and inhibitory, whereas the d subunits, which are endogenous molecules of calmodulin, are stimulatory. It is through allosteric sites on these regulatory subunits that PhK integrates metabolic (ADP), hormonal (cAMP and Ca 2+ ), and neural (Ca 2+ ) signals to control the rate of glycogen breakdown through large increases in the kinase activity of its catalytic g subunits. Ca 2+ ions presumably exert their stimulatory effect through binding to the d subunits, which in muscle couples contraction with energy production to supply

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Section: Discussionmentioning

confidence: 99%

Physicochemical changes in phosphorylase kinase associated with its activation

2008

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“…Anti-calmodulin serum (clone CaM85) was from Zymed Laboratories, Inc. (South San Francisco, CA). The polyclonal antibody to the PhK complex that detects α, β, and γ subunits was raised in guinea pigs (23). The goat polyclonal antibody against GAPDH was from Imgenex Corp. (San Diego, CA).…”

Section: Electrophoresis and Immunoblottingmentioning

confidence: 99%

The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde-3-phosphate Dehydrogenase

et al. 2008

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Skeletal muscle phosphorylase kinase (PhK) is an (αβγδ) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GPb) in a Ca 2+ -dependent reaction that couples muscle contraction with glycogen breakdown. GPb is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [γ-32 P]ATP with or without Ca 2+ and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca 2+ -dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 0 .45 µM) PhK's conversion of GPb. When a short synthetic peptide substrate was substituted for GPb, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the γ subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory β subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the β subunit, specifically an αγδ subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GPb. Moreover, GAPDH had no effect on the conversion of GPb by the αγδ subcomplex. The interactions described herein between the β subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.Phosphorylase kinase (ATP:phosphorylase b phosphotransferase, EC 2.7.1.38), hereafter termed PhK, 1 is in fast-twitch skeletal muscle a hexadecameric complex with a subunit composition of (αβγδ) 4 and a mass of 1.3 × 10 6 Da (reviewed in ref 1). The activity of its catalytic γ subunit (44.7 kDa) is controlled by its regulatory α (138.4 kDa), β (125.2 kDa),

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“…The anti-CaM mAb (clone CaM85) was from Zymed Laboratories, Inc. (South San Francisco, CA). Guinea pig polyclonal antibodies (pAbs) against the α, β , and γ subunits of PhK were described previously (8). The penta-His mouse mAb was from S Prime Inc. (Gaithersburg, MD).…”

Section: Methodsmentioning

confidence: 99%

“…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 α γδ 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.…”

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

et al. 2009

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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.

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Baculovirus-Mediated Overexpression of the Phosphorylase b Kinase Holoenzyme and αγδ and γδ Subcomplexes

Cited by 5 publications

References 29 publications

Physicochemical changes in phosphorylase kinase associated with its activation

Physicochemical changes in phosphorylase kinase associated with its activation

The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde-3-phosphate Dehydrogenase

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

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