Phosphorylase kinase, regulation

An example for the reversible association of activator proteins with an enzyme is the Ca -calmodulin dependent enzymes. Calmodulin is a Ca -binding protein which can activate target enzymes, e.g. phosphorylase kinase (see 6.7.1 and 7.4) in its Ca -boimd form. Another example for activating proteins is the cyclins (see chapter 14). The cyc-lins are activators of protein kinases that regulate the cell cycle. 

Fig. 7.18. Regulation of glycogen metabolism in muscle. Phosphorylase kinase stands at the center of regulation of glycogen metabolism. Phosphorylase kinase may exist in an active, phosphorylated form and an inactive, unphosphorylated form. Phosphorylation of phosphorylase kinase is triggered by hormonal signals (e.g. adrenahne) and takes place via an activation of protein kinase A in the cAMP pathway. In the absence of hormonal stimulation, phosphorylase kinase can also be activated by an increase in cytosolic Ca. The active phosphorylase kinase stimulates glycogen degradation and inhibits glycogen synthesis, in that, on the one side, it activates glycogen phosphorylase by phosphorylation, and on the other side, it inactivates glycogen synthase by phosphorylation.

Fig. 7.19. Subunit structure and regulation of phosphorylase kinase of muscle. Phosphorylase kinase is - according to the excitation state of the muscle - regulated by two pathways. On nervous stimulation of the muscle, voltage-controlled Ca channels are opened, the cytosolic Ca concentration increases and Ca binds to calmoduhn, activating phosphorylase kinase. In resting muscle, activation of phosphorylase kinase is triggered by a hormonal signal. A hormonal signal initiates phosphorylation of the a and P subunits of phosphorylase kinase. In the phosphorylated form, Ca binding affinity of the calmodulin subunit (8) is strongly increased and activation is also possible at low Ca concentrations.

The examples of phosphorylase kinase and protein phosphatase I illustrate some important principles of regulation of enzyme activity by phosphorylation and dephosphorylation events. They clearly indicate how different signal transduction paths can meet in key reactions of metabolism, how signals can be coordinated with one another and how common components of a regulation network can be activated by different signals. The following principles are highlighted ... 

A protein kinase or protein phosphatase may be regulated by different signal transduction pathways. Thus, different external stimuli may influence the phosphorylation status of a protein. This differential stimulation may be mediated by the subunits of the enzyme, for example. For phosphorylase kinase, a Ca signal is registered by the Ca T calmodulin subunit whereas a cAMP protein kinase A signal is picked up in the form of a phosphorylation of the a and P subunits. Which of the signals comes into play depends on the current metabolic situation. 

FIGURE 6-31 Regulation of glycogen phosphorylase activity by covalent modification. In the more active form of the enzyme, phosphorylase a, specific Ser residues, one on each subunit, are phosphorylated. Phosphorylase a is converted to the less active phosphorylase b by enzymatic loss of these phosphoryl groups, promoted by phosphorylase phosphatase. Phosphorylase b can be reconverted (reactivated) to phosphorylase a by the action of phosphorylase kinase. 

Phosphorylase kinase, one of the Ser/Thr-kinases, is oligomeric with the stoichiometry (a/3yS)4.80> The y-subunit of the enzyme is homologous to other protein kinases and possesses protein kinase activity when separated from other subunits.811 Phosphorylase kinase was more effectively inactivated by AP3-PL and AP4-PL than PLP and AP2-PL. Ca2+ and Mg2+, activators for this enzyme, enhanced the degree of inactivation by all the pyridoxal compounds. Inactivation by AP3-PL and AP4-PL was markedly protected by adenylyl, /3,y-imidodiphosphate, a nonhydrolyzable analogue of ATP, and ADP. Because the a- and /3-subunits have regulatory ATP-binding sites and the kinase activity is regulated by these subunits,821 the incorporation of AP3-PL into the y-subunit and... 

Some protein kinases are cAMP-independent and respond instead to increased free Ca2+ levels. Some of these kinases contain the calcium-binding subunit calmodulin. Binding of Ca2+ to calmodulin induces an allosteric activation analogous to the activation of protein kinases by cAMP. Some protein kinases, such as phosphorylase kinase, are regulated by both cAMP and Ca2+. 

Figure 18.14 Glycogen biosynthesis and degradation regulation. cAMP activates cAMP-dependent protein kinases. They cause the phosphorylation of glycogen synthase (inactivation), phosphorylase kinase (activation), and the inhibitory protein. The last inhibits phosphoprotein phosphatase. Activated phosphorylase kinase causes the phosphorylation of phosphorylase b, thus activating it to phosphorylase a. Phosphoprotein phosphatase is inhibited by the phosphorylated inhibitor protein. Such inhibition is released when the inhibitor protein is dephosphorylated. The phosphatase then reactivates glycogen synthase and inactivates phosphorylase kinase and phosphorylase a.

Phosphorylase kinase is one of the best characterized enzyme systems to illustrate the role of calcium ions in regulation of intermediary metabolism. Phosphorylase kinase is composed of four different subunits termed a (Mr 145000), /3 (MT 128000), y (A/r 45000) and 5 (Mr 17000) and has the structure (a/3y8)A [106]. Only one of its four subunits actually catalyses the phosphorylation reaction the other three subunits are regulatory and enable the enzyme complex to be activated both by calcium and cyclic AMP. The y subunit carries the catalytic activity the 8 subunit is the calcium binding protein calmodulin and is responsible for the calcium dependence of the enzyme. The a and /3 subunits are the targets for cyclic-AMP mediated regulation, both being phosphorylated by the cyclic-AMP dependent protein kinase. Calmodulin appears to interact with phosphorylase kinase in a different manner from other enzymes, since it is an integral component of the enzyme. Phosphorylase kinase has an absolute requirement for calcium, and is inactive in its absence. 

Since phosphorylase kinase not only activates phosphorylase, but also phospho-rylates glycogen synthase thereby decreasing its activity, the regulation of phosphorylase kinase by calcium may also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction. 

How could the activities of the kinase and phosphorylase be regulated so as to control the entry of pyruvate into the tricarboxylic acid cycle ... 

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