In the cascade activation of glycogen utilization leading to energy production in mammalian skeletal muscle, phosphorylase kinase (PhK) phosphorylates and activates glycogen phosphorylase, which then degrades glycogen. The ( alpha-beta-gamma-delta)4 PhK complex is among the largest and most complex enzymes known, with 90% of its 1.3 x 10(6) Da mass involved in its regulation (i.e., the integration of activating signals). The activity of PhK, catalyzed by its gamma subunit, is markedly enhanced by multiple metabolic, hormonal and neural stimuli, which it integrates through allosteric sites on its three regulatory subunits, alpha, beta and delta. Ca2+ is an obligatory activator, in that PhK's other activators have no effect in its absence, but further activate in its presence. The simultaneous activation of PhK by diverse biological signals allows for the tight control of glycogenolysis and subsequent energy production;and in skeletal muscle, activation of PhK by Ca2+ couples contraction with energy production to sustain contraction. Moreover, a regulatory region of PhK's catalytic subunit shares high sequence identity with the key functional region of the troponin-actin Ca2+-dependent regulatory system of muscle contraction, suggesting co-evolution of the regulation of contraction and energy production from glycogen. Our long term goal is to elucidate the changes in intersubunit interactions that lead to activation of PhK in response to different biological signals and the interplay between changes induced by the obligatory Ca2+ versus PhK's other activators. The proposed project consists of two broad aims related to the effects of activators on PhK. (1) We will characterize activator induced structural changes in PhK from the subunit level to the overall complex by employing a large variety of chemical, biochemical and biophysical techniques. (2) Through baculovirus (BV)-mediated expression of recombinant complexes, we will evaluate the function of residues and regions that are thought to participate in PhK's communication network. This will mark the first ever direct structure-function studies on this massive enzyme complex. The BV-expression system will also allow elucidation of the underlying reasons for human glycogen storage diseases caused by single missense mutations in PhK's large and regulatory subunits, which in the case of alpha can lead to different phenotypes depending on where in the subunit that mutation occurs.

Public Health Relevance

Glycogen is the storage form of glucose in mammals and can be readily mobilized, a process that is particularly important in muscle. The key enzyme for regulating its mobilization is phosphorylase kinase (PhK), which is one of the most complex regulatory enzymes known, but is still poorly understood. Glycogen storage disease, or glycogenosis, a common inborn error of metabolism, is most often caused by mutations in the regulatory subunits of PhK. This work will elucidate underlying mechanisms for the regulation of normal PhK and for the cause of PhK deficiency and glycogenosis in people harboring mutations in its regulatory subunits.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK032953-30
Application #
8462956
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Sechi, Salvatore
Project Start
1986-09-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
30
Fiscal Year
2013
Total Cost
$413,255
Indirect Cost
$137,752
Name
University of Kansas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
016060860
City
Kansas City
State
KS
Country
United States
Zip Code
66160
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Jeyasingham, Marina D; Artigues, Antonio; Nadeau, Owen W et al. (2008) Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle. J Mol Biol 377:623-9
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Nadeau, Owen W; Gogol, Edward P; Carlson, Gerald M (2005) Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase. Protein Sci 14:914-20
Priddy, Timothy S; MacDonald, Brian A; Heller, William T et al. (2005) Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering. Protein Sci 14:1039-48
Rice, Nancy A; Nadeau, Owen W; Yang, Qing et al. (2002) The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions. J Biol Chem 277:14681-7
Venien-Bryan, Catherine; Lowe, Edward M; Boisset, Nicolas et al. (2002) Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b. Structure 10:33-41
Nadeau, Owen W; Carlson, Gerald M; Gogol, Edward P (2002) A Ca(2+)-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy. Structure 10:23-32

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