Molecular Biology: Protein Structure and Function - Stepanov V.M. 2005

Post-translational Protein Modification
Protein Phosphorylation. Phosphoproteins

Proteins phosphorylate specific Enzymes known as protein Kinases, which catalyze The transfer of the terminal phosphate of ATP to the hydroxyl groups of Serine or Threonine residues. Tyrosine phosphorylation occurs significantly less frequently—with phosphotyrosine accounting for only about 0.1% of the total phosphoserine and phosphothreonine content—yet The Physiological Role of this modification is profound.

Class="center">

The phosphoester bonds of Phosphoproteins can be hydrolyzed by specialized enzymes called phosphoprotein Phosphatases. Thus, phosphorylation is a reversible process. Over its lifespan, a protein may repeatedly transition between phosphorylated and dephosphorylated states. This crucially important feature of phosphorylation—the reversibility of the modification—underpins its role as a mechanism for regulating The activity of individual proteins, and even entire groups of proteins, in response to changing intra- and extracellular stimuli.

The Role of phosphorylation in regulating phosphorylase is well understood. Phosphorylase is the enzyme that catalyzes the phosphorolysis of the storage polysaccharide Glycogen to yield glucose-1-phosphate, which is subsequently utilized in METABOLISM/26.html">Energy Metabolism. The reverse reaction—the incorporation of a glucose residue from glucose-1-phosphate into glycogen—is catalyzed by glycogen synthase. The concerted action of these two enzymes drives the cycle of transformations shown below:

Such a cycle requires precise control: the alternating activation of phosphorylase or synthase activity coupled with the downregulation of the opposing pathway. Without this regulation, the cycle would be rendered futile. Indeed, the activity of Glycogen phosphorylase is controlled by a multitude of factors, one of which is the phosphorylation and dephosphorylation of the enzyme itself. Unphosphorylated phosphorylase b is virtually inactive, but it is converted into its active form—phosphorylase a—upon the phosphorylation of a single serine residue (Ser-14) by the appropriate protein kinase. How can one account for the activation of an enzyme whose subunit consists of approximately 400 amino acid residues As a result of what appears to be a minor covalent modification?

The serine residue subject to phosphorylation is located at the contact interface between two phosphorylase subunits, i.e., in a region of relatively weak interactions that is exceptionally prone to conformational rearrangement even upon minor changes in Covalent Structure. The attachment of a phosphate group to Ser-14 induces an electrostatic interaction and The formation of two simultaneous Hydrogen Bonds between the phosphoester group of phosphoserine and the guanidino group of an Arg-69 residue, which belongs to the opposing subunit and is similarly localized on the contact surface:

This alters the network of non-covalent bonds at the subunit contact interface, and the effect is transmitted directly to the catalytic site of the enzyme. As a result, the peptide loop that shields the active center in phosphorylase b swings away, granting substrate access and thereby activating the enzyme.

The Cleavage of the phosphate group by phosphoprotein phosphatase reconverts phosphoserine-14 back to serine, restoring The system of non-covalent interactions characteristic of inactive phosphorylase b at the subunit boundary. The peptide loop then returns to its original position, blocking the catalytic center.

Thus, the profound functional consequences of phosphorylation (and dephosphorylation) stem from the fact that these reactions take place within a relatively labile zone of the Cell/13.html">Protein Structure—namely, the contact region between subunits (as in the case discussed) or domains. Interestingly, the regulatory effect of AMP on phosphorylase is based on a similar principle: adenosine monophosphate forms a stable, non-covalent complex with phosphorylase b by binding at the subunit interface. Its phosphate group interacts with two guanidino groups of Arginine residues, triggering a cascade of structural rearrangements within the structurally labile zone of the enzyme—analogous to The Effect of Ser-14 phosphorylation—which once again exposes the catalytic center. The result is the activation of phosphorylase b, an enzyme that is practically inactive in the absence of AMP. The difference lies in the fact that phosphorylation activates the enzyme by converting it into phosphorylase a independently of the intracellular concentration of the effector (in this case, AMP) and, consequently, to a certain extent independently of The Cell's metabolic status.

As noted previously, the transfer of a phosphate from ATP to the enzyme is catalyzed by a protein kinase. The activity of this enzyme is, in turn, controlled by and dependent on the intracellular concentration of a so-called "second messenger"—cAMP. The cAMP-dependent protein kinase is composed of dissimilar subunits: a regulatory R-subunit capable of binding the effector (cAMP), and a catalytic C-subunit. The regulatory subunit acts as a protein kinase inhibitor; when complexed with it, the catalytic subunit is devoid of activity. Upon binding cAMP via non-covalent interactions, the regulatory subunit loses its ability to maintain The quaternary structure. As a result, the catalytic subunit is released and becomes capable of phosphorylating target proteins.

A decrease in the intracellular concentration of free cAMP causes the excess synthesized regulatory subunit to rebind to the catalytic subunit, leading to Enzyme Inhibition. This establishes a dynamic equilibrium:

Thus, the concentration of cAMP governs the activity of protein kinase, which in turn regulates glycogen phosphorylase activity and, ultimately, the mobilization of energy stored by the cell in the form of glycogen. The level of cAMP is determined by the relative activities of adenylate cyclase—the enzyme that converts ATP into cAMP—and phosphodiesterase, which hydrolyzes cAMP to AMP. Adenylate cyclase activity depends on a variety of external factors, among which hormonal control is particularly crucial. Therefore, the phosphorylation of phosphorylase b and its conversion into the active phosphorylase a enzyme is the result of a meticulously fine-tuned cascade of enzymatic reactions. As previously mentioned, phosphorylation is not the sole mechanism for modulating phosphorylase activity.

As evident from the cycle discussed above, the activation of phosphorylase must be accompanied by a decrease in glycogen synthase activity. This enzyme also exists in two forms, yet in this case, the active form is unphosphorylated. Upon phosphorylation by the appropriate protein kinase, it is converted into inactive glycogen synthase.

Consequently, the aforementioned cycle of glycogen synthesis and phosphorolysis must be expanded to include a rather complex network of interconversions between the active and inactive forms of the enzymes catalyzing these processes:

It must be kept in mind that even this scheme is a significant oversimplification. It has been established that protein kinase activity can be regulated by calmodulin—a calcium-ion-binding protein—and is therefore also dependent on the concentration of this second messenger within the cell.

Similarly, the activity of a broad range of other metabolic enzymes—most notably fatty acid synthase and Pyruvate dehydrogenase—is controlled through phosphorylation and dephosphorylation. It is important to emphasize that phosphorylation and dephosphorylation reactions differ fundamentally from allosteric enzyme regulation: they are not directly dependent on the biochemical status of the cell and can therefore be utilized to alter activity in response to extracellular signals, making them well-suited for systemic Regulation at the organismal level. Other protein kinases capable of phosphorylating serine or threonine residues in proteins are also known, including those independent of cAMP or regulated by cGMP and diacylglycerol.

The Selection of the specific serine residue targeted for phosphorylation may depend on the surrounding Amino Acid Sequence. For instance, bovine cAMP-dependent protein kinase phosphorylates the serine hydroxyl group within the sequence

Arg—Arg—Ala—Ser—Val (Leu),

where the adjacent arginine residues appear to serve a directing role for the enzyme.

Tyrosine phosphorylation, catalyzed by specific protein kinases (tyrosine kinases), occurs far less frequently than the phosphorylation of Serine and threonine residues—with phosphotyrosine accounting for roughly 0.1% of all protein-bound phosphate. Nonetheless, the Biological Significance of this reaction is immense, as it plays a central role in signal Transduction across biological systems.

For instance, upon binding to its receptor—a transmembrane protein—Insulin triggers the activation of its cytoplasmic domain, which exhibits tyrosine kinase activity. Tyrosine kinase activity is also induced by a variety of growth factors, which are proteins that trigger Cell Division via a mechanism analogous to that described for insulin. The receptors for these growth factors are large transmembrane proteins that bind their ligands on the outer surface of the membrane. The Formation of the factor-receptor complex transmits the signal across the transmembrane region of the receptor to its cytoplasmic tyrosine kinase domain. The thus-activated tyrosine kinase then modifies intracellular proteins, propagating the signal downstream.

Tyrosine kinases phosphorylate tyrosine residues in certain intracellular proteins involved in the complex regulatory system of cell division, which also entails the phosphorylation of proteins at serine and threonine residues. Impairment of this highly sophisticated regulatory network can lead to uncontrolled cell division and tumor growth. Characteristically, oncoproteins—which are structurally similar to normal animal cell proteins yet induce cellular transformation into a tumor—frequently turn out to be tyrosine kinases.