Kinases
Phosphorylation has been defined. The enzymes that catalyze this phosphorylation are called kinases. Therefore, phosphorylation can be described as the process where a protein kinase, a catalytic enzyme, attaches a phosphate group to a substrate protein, modifying the function of another protein. In the phosphorylation process, there are three key components: a donor that provides the phosphate, the substrate protein that receives the phosphate, and the kinase enzyme that mediates the reaction. However, we must add one more very important element: the cofactor. This is typically a metal ion, represented by magnesium ions (Mg²⁺). This metal ion plays a crucial role in facilitating the transfer of phosphate and stabilizing ATP binding. The phosphate donor, of course, is ATP within the cell. One reason phosphorylation is a popular method for protein modification is likely due to the abundant supply of phosphate through ATP. Mitochondria in cells diligently produce ATP through cellular respiration, so ATP, which carries three phosphate groups, is always plentiful, making it easy to obtain the materials needed for phosphorylation.
For reference, it is said that the human genome contains over 500 genes responsible for producing these protein kinases and more than 200 genes for the phosphatases that counteract them. About 3.5% of human genes are used to directly regulate protein phosphorylation, and it is estimated that approximately 30% of proteins undergo phosphorylation.[1] It is also estimated that there are about 13,000 proteins in the human body that can be phosphorylated.[2] Considering these figures, we can gauge the significance of the pathological aspects related to kinases.
Classification of kinases
There are various ways to classify kinases. First, based on the target substrate, they can be broadly categorized into carbohydrate kinases, lipid kinases, and protein kinases. However, this article will focus on protein kinases. They can also be divided according to their function, specifically whether they act on a single substrate or multiple substrates. In this article, we will classify them based on three representative amino acids that undergo phosphorylation: serine, threonine, and tyrosine. The kinases that phosphorylate serine and threonine are actually very similar and differ very little, so they are typically grouped together as serine/threonine kinases. Additionally, there are tyrosine kinases that act on tyrosine. Moreover, there are dual specificity kinases that can phosphorylate all three amino acids. Mitogen-activated protein kinases (MEKs) are a representative example of this. While it is possible to classify them in more detail, they are generally categorized into these three major groups.
First, serine/threonine kinases (STKs) represent the largest family among protein kinases. Out of over 500 kinases, at least 350 belong to this category. In fact, I initially intended to summarize and introduce a few key kinases that phosphorylate serine/threonine sites, but I stopped midway due to the sheer volume of information. It was so extensive that it was difficult to decide which ones to highlight. Moreover, I also planned to introduce the important intracellular signaling pathways involved with STKs, but I abandoned this idea as well because there were too many complex pathways, making it challenging to organize the information in a clear and understandable way. The reason I wanted to address kinases in detail was precisely due to their significance and diversity across numerous pathways, so it seems more appropriate to cover the various pathways in detail later on.
Tyrosine kinases are particularly important in receptor tyrosine kinases, which are the binding mechanisms for most growth factors and immune-related signaling molecules. We will examine the initial binding point and method of interaction between cells and external signals, which is the first step in the intracellular signaling system, in more detail in a later section.
Structure and Process of Kinases by PKA (Protein Kinase A)
How is a kinase structured internally? The binding sites where the enzyme interacts with ATP and the substrate, along with the catalytic site where the catalytic activity occurs, are collectively referred to as the active site. Most of the remaining portions of the enzyme, excluding these active sites, are composed of scaffold proteins that help maintain the overall structure of the enzyme and allow the kinase to position itself closer to the substrate.
It is said that kinases identify the sites to be phosphorylated based on the amino acid sequence. The substrate proteins, made up of numerous amino acids, contain countless serine, threonine, and tyrosine residues. To determine which of these residues should be phosphorylated, kinases read the amino acid sequence surrounding the residues. In other words, they locate the phosphorylation target sites by examining the amino acid sequence around the substrate protein residues.
Although the amino acid sequences of various kinases in the human body are diverse, there are certain amino acids that are conserved across all kinases. Let's take protein kinase A (PKA), which was discovered first and has been the most extensively studied, as an example to glimpse the structure and reaction process of kinases.[3] PKA is a serine/threonine kinase (STK).
For catalytic activity to occur properly, the substrate must fit well into the active site of the kinase. Therefore, the structure is essential. Additionally, once the substrate fits in, it must remain in the active site long enough for the catalytic reaction to take place. Keeping this in mind, let's examine the structure of the kinase. Kinases are composed of two lobes (bi-lobe), which contain regions that bind ATP and parts that interact with the target protein substrate. There is also a small linker region that connects these two parts, functioning like a hinge that allows the two lobes to open and close according to the catalytic reaction phase.
Between the two lobes, there is a space where the catalytic action of transferring and attaching phosphate occurs. This pocket-like area contains a glycine-rich loop that helps the kinase bind well to ATP (preventing it from escaping?). Additionally, the aspartate (Asp) in the active site of the kinase plays a very important role in interacting with divalent magnesium ions (Mg²⁺). Specifically, Mg²⁺ not only accurately positions the γ phosphate group of ATP (the outermost phosphate group, as shown in the diagram above) for direct transfer to the substrate, but it also shields the charge of the γ phosphate group, reducing electrostatic repulsion with the substrate's hydroxyl group. Lysine (Lys) also interacts with the α and β phosphates of ATP, providing additional stability. Furthermore, Asp forms hydrogen bonds with the substrate's hydroxyl group, ensuring proximity and facilitating the substrate's hydroxyl group to be in a favorable position for reaction with ATP's γ phosphate. The diagram above illustrates the interactions of certain amino acid residues that are crucial for kinase action with the various participants in protein phosphorylation.
Phosphorylation and Dephosphorylation: Constant Checks & Balances
In humans, it is estimated that there are about 13,000 different proteins that can be phosphorylated. While kinases are the enzymes that attach phosphate groups, there are also enzymes that remove them. Therefore, phosphorylation is a reversible reaction. The process of removing phosphate from organic compounds is called dephosphorylation, which involves phosphatases that catalyze the breakdown using water (H₂O). This smooth flexibility, where phosphate groups can be added and then removed, forms the basis for kinases to act like switches that turn protein activity on and off. By adding phosphate, enzymes can increase their production, and if the amount becomes excessive, phosphate can be removed to stop the process, thereby regulating the quantity.
In the intricately unfolding intracellular signaling processes, the phosphorylation of various phosphoproteins within specific pathways is controlled, leading to a variety of physiological effects. By adding and removing phosphates, kinases awaken dormant proteins at each stage to activate the next ones. What is particularly remarkable about the process of phosphorylation is that there is always a counterpart that can check, regulate, and control it. This check and balance ensure cellular homeostasis and normal function. Unfortunately, if this process malfunctions for any reason, leading to a loss of balance, it can result in various diseases, with cancer being a prominent example. Balance is essentially a mechanism to prevent the excessive expression of specific genes, and cancer occurs when cells that should have undergone apoptosis continue to proliferate uncontrollably. Therefore, kinases are highly relevant in the context of cancer as well.
Intracellular signaling pathways and kinases
Signaling pathways are incredibly complex, overlapping, and mutually influencing, evoking the phrase "orderly chaos." To achieve a single cellular response, these pathways may start from different types of receptor binding and proceed along distinct routes. Similar kinases from the same kinase family can diverge into different pathways, or different kinases can phosphorylate the same protein kinase site. For those studying this field, the complexity can be quite perplexing, yet it also evokes a sense of wonder. The seamless process in which numerous proteins are phosphorylated by kinases, leading to the next stages of signaling, is remarkably harmonious.
When all these pathways progress smoothly, cells maintain homeostasis and function normally, contributing to a stable and uneventful day for us. There are mechanisms in place at every step to ensure that no proteins run amok, maintaining balance throughout the pathways. All of these elements constitute a vast mechanism that allows us to function healthily. A significant part of this intricate system is played by kinases. For cells to grow, differentiate, and, when necessary, undergo apoptosis to survive healthily, checks and balances must be in place. Cancer, in contrast, arises when this order is violated, leading to unchecked growth and proliferation.
To gain a more clear understanding of these cellular functions and their pathways, I now intend to explore the intracellular signaling processes in great detail, returning to the original starting point.
[References]
[1] A Mechanism for the Evolution of Phosphorylation Sites
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220604/
[2] Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466708/
[3] Ca(2+)/calmodulin-dependent protein kinases
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617042/pdf/18_2008_Article_8086.pdf
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