PHK

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Introductions

Phosphorylase kinase (PhK) coordinates hormonal and neuronal signaling to initiate glycogenolysis. PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), leading to the formation of active glycogen phosphorylase a.

Structure

PhK is one of the largest and most complex protein kinases. Autophosphorylation or phosphorylation of these subunits by cyclic adenosine monophosphatase-dependent kinases in response to hormonal stimulation leads to kinase activation. ADP binds to the β subunit with high affinity and stimulates activity. Ca²⁺ affects neuron-stimulating ions that bind to the intrinsic calmodulin (δ) subunit (16.7 kDa, 148 amino acids), thereby binding muscle contraction to energy production. PhK differs from most other calmodulin-regulated enzymes in that the δ-calmodulin subunit is tightly associated in the holoenzyme in the absence of calcium. It consists of a kinase structural domain (residues 20-276) and a regulatory calmodulin-binding autoinhibitory structural domain (residues 298-396). phK is one of several kinases that do not require phosphorylation on the kinase activation fragment to be active.

Function

PhK is a key enzyme in the control of glycogenolysis. Intracellular glycogen reserves are primarily used to maintain glucose homeostasis during fasting and as a source of energy for muscle contraction. PhK integrates signals from different signaling pathways - hormonal messengers (epinephrine), neuronal stimulation (Ca²⁺), and metabolic signals (e.g., adenosine diphosphatase [ADP] levels) - to produce rapid mobilization of glycogen reserves. PhK phosphorylates inactive glycogen phosphorylase b (GPb) on a single serine Ser14, converting it to active phosphorylase a (GPa). Active phosphorylases catalyze glycogen phosphorylation to release glucose-1-phosphate. All eukaryotic protein kinases share a common catalytic core that catalyzes the transfer of γ-phosphate from a nucleoside triphosphate donor to the side chain hydroxyl group of a serine, threonine or tyrosine in a substrate protein, but each kinase recognizes only a specific substrate and each is regulated by a different mechanism.

Activation

The activation of PhK is complex and has several different control layers. In vitro pH is a further regulator. Inactive PhK measured in the presence of calcium exhibits low activity at pH 6.8. Activity increases significantly at pH 8.2, but still requires the presence of calcium. Activation of PhK by phosphorylation or limited protein hydrolysis resulted in increased activity at pH 6.8, but little change in activity at pH 8.2.

Mechanism

The precise details of the catalytic mechanism of PhK are still being investigated. There are great difficulties in studying the finer details of the structure and mechanism of PhK with a high degree of complexity. At the active site, there is significant homology between PhK and other so-called P-loop protein kinases such as protein kinase A (PKA, cAMP-dependent kinase). In contrast to these other proteins that normally require phosphorylation of serine or tyrosine residues in the catalytic site in order to be active, the catalytic γ subunit of PhK is constitutively active due to the presence of the negatively charged glutamate residue Glu-182. Structural and biochemical data suggest that one possible mechanism of action of PhK phosphorylated glycogen phosphorylase involves the direct transfer of phosphate from adenosine triphosphate (ATP) to the substrate serine.