Introduction to Phosphatases and Phosphatase Inhibitor Cocktails

Phosphorylation-dephosphorylation, or the addition and removal of phosphate groups of biomolecules, are key determinants of physiology. Because of this, studying phosphatases is very important for bioscience research to understand the various cellular processes they regulate via catalyzing dephosphorylation reactions.

Phosphatases are a class of enzymes that catalyze the removal of phosphate groups from its substrates. Classified based on their substrates, many different types of phosphatases occur in biological systems.

Phosphatase inhibitors, on the other hand, are compounds that inhibit activity of phosphatase enzymes.

Scientists use phosphatase inhibitors in experiments to avoid unwanted or accidental dephosphorylation of their target biomolecules during experimental steps, which might lead to erroneous results.

Often in bioscience research, multiple phosphatase inhibitors are used together in the same experiment to inhibit a wide range of different phosphatases at the same time. This is like a cocktail drink that contains several different alcohols, and therefore a mix of phosphatases is called a phosphatase inhibitor cocktail.

In this article, we will look at the basic biochemistry of phosphatases. Since protein phosphorylation and dephosphorylation events are especially important physiological signals, we will take a deeper dive into protein phosphatases, enzymes that dephosphorylate protein substrates.

Also, we will discuss why protein phosphatases have a special significance in some experimental lab work, and why phosphatase inhibitor cocktails are used in those experiments.

Article Table of Contents:

What are Phosphatases?

What are the different types of phosphatases?

Carbohydrate phosphatases

Nucleotidases

Lipid phosphatases

Types of protein phosphatases

What are phosphatase inhibitor cocktails?

Why are phosphatase inhibitor cocktails used in experiments?

References

What are Phosphatases?

Phosphatases are enzymes that catalyze the dephosphorylation of their substrates which are proteins, nucleotides, and some lipids and carbohydrates.

Dephosphorylation is the removal of a phosphate group from a molecule by a hydrolysis reaction. Because of this, phosphatases fall under the enzyme category hydrolases.

Kinases are enzymes whose function is just opposite to what phosphatases do.

Kinases phosphorylate or add phosphate groups to their substrates.

Figure 1 illustrates the functions of kinase and phosphatase enzymes.

Figure 1. Phosphatase kinase reaction. Kinases catalyze the reaction where a phosphate group is transferred from an ATP to the substrate molecule. The phosphate group is then added to the substrate. Phosphatase enzymes removes phosphate groups from the substrate.

What are the 4 different types of phosphatases?

Different phosphatases, classified based on their substrate specificities, are:

• Protein phosphatases: dephosphorylate specific amino acids that may be phosphorylated in a protein.
• Carbohydrate phosphatases: dephosphorylate sugar phosphates.
• Nucleotidases: break down nucleotides into a nucleoside and phosphate.
• Lipid phosphatases: dephosphorylate selected lipids such as phosphatidylinositol 3, 4, 5-triphosphate.

Among these, protein phosphatases are pivotally important in physiological relevance.

Here are brief notes on the three other types of phosphatases.

Carbohydrate phosphatases

Carbohydrate phosphatases remove phosphate groups from phosphorylated sugars. For example, glucose 6-phosphatase acts on glucose 6-phosphate to produce glucose.

These dephosphorylation reactions, catalyzed by carbohydrate phosphatase enzymes, have huge significance in metabolic pathways such as gluconeogenesis.

Nucleotidases

Nucleotidases are enzymes that catalyze the breakdown of nucleotides into the corresponding nucleosides and phosphate groups. For this reason, nucleotidases play an important role in nucleic acid metabolism.

An example is adenosine monophosphate (AMP), a nucleotide, is dephosphorylated by a nucleotidase to yield adenosine, a nucleoside, and a phosphate.

Lipid phosphatases

Lipid phosphatases dephosphorylate phospholipids. Phospholipids, for example phosphatidylinositol 3,4,5-triphosphate, play important roles in cell signaling.

As a result, lipid phosphatases such as Myotubularin, regulate signal transduction pathways involved in many aspects of physiology including diseases such as myotubular myopathy.

Types of protein phosphatases

Protein phosphatases remove phosphates from amino acids that can be phosphorylated in a protein – serine, threonine and tyrosine.

Depending on which amino acids a protein phosphatase can dephosphorylate, it is classified as:

• Tyrosine phosphatase
• Serine/ threonine phosphatase
• Dual specificity phosphatase that can dephosphorylate both phosphor-tyrosines and phosphoserine/ phosphothreonines.

Recently, histidine has also been reported to be phosphorylated in some physiological situations; histidine phosphatases dephosphorylate these residues, making them a fourth category of protein phosphatases.

Serine and threonine have similar structures. Notably, both of these amino acids have a hydroxyl (-OH) group that can get phosphorylated.

Figure 2 depicts how phosphoserine and phosphothreonine gets dephosphorylated by serine/ threonine phosphatases to yield a serine and a threonine respectively.

Figure 2. Serine/Threonine phosphatase mechanism of action

Figure 3 shows the dephosphorylation of a phosphorylated tyrosine (phosphotyrosine) by a tyrosine phosphatase.

Figure 3. Tyrosine phosphatase mechanism of reaction

Like serine and threonine, tyrosine also has a hydroxyl group that can get phosphorylated.

Significance of protein phosphatases

Proteins that have the necessary amino acids in its sequence for getting phosphorylated, may or may not be phosphorylated at a given point in time.

Whether such a protein is phosphorylated or partially phosphorylated or not phosphorylated, determines its phosphorylation status.

Phosphorylation status of proteins regulate their interactions with other biomolecules. This ultimately controls multiple aspects of physiology such as cell signaling, enzyme kinetics, gene expression, protein degradation etc.

Since protein kinases and protein phosphatases catalyze protein phosphorylation and dephosphorylation respectively, these enzymes regulate these physiological events.

Figure 4 is an example of how important a protein phosphatase can be. In this case, improper synthesis or function of protein phosphatases can lead to Alzheimer’s disease.

Figure 4. Represents the importance of protein phosphatase in a biological system. While Tau proteins in a healthy brain are not hyperphosphorylated, in the brain of an Alzheimer's patient, Tau proteins are often found in a hyperphosphorylated state. This is because, in normal (healthy) conditions, there are phosphatase enzymes (Phosphatase 2A and 2B) which dephosphorylate Tau and prevent the protein from existing as hyperphosphorylated. In Alzheimer's disease, often there are mutations in these phosphatase enzymes. This ultimately results in Tau protein being hyperphorylated in brains of Alzheimer's patients, which is an important attribute to the disease pathophysiology.

Here are some fun facts that highlight the significance of protein phosphorylation-dephosphorylation reactions in biosystems such as cells and tissues:

• Phosphorylation-dephosphorylation of proteins are reversible, post-translational modifications.
• Around 33% of proteins in a cell are phosphorylated at a time. These proteins constitute the phosphoproteome of that cell.
• Some proteins might be phosphorylated at multiple amino acid sites.
• There may be multiple amino acid residues in a protein suitable for phosphorylation. However, only a subset of these may be phosphorylated at a certain physiological time.
• The same protein may have a different phosphorylation-dephosphorylation profile at different time points as well as in different physiological environments.
• Many diseases are solely caused due to altered phosphorylation status of proteins.

As you can see, studying protein phosphorylation-dephosphorylation events are actually very important in research.

The first step when studying the phosphorylation-dephosphorylation physiology of any protein, is to ensure that during cell lysis, the protein is extracted in its proper phosphorylated or dephosphorylated form, as it actually exists in the physiological system that is being studied.

In other words, during their extraction, the phosphorylation-dephosphorylation status of the proteins, especially the one under study, need to be fixed or frozen in the exact same way as they exist in the actual physiological context.

Here is an example to help you understand this.

Say, a protein exists as phosphorylated in the actual biological system being studied. For example, the hyperphosphorylated Tau protein in Alzheimer’s brain tissue.

Now, during extraction of this protein, it is extremely important that these phosphorylations of the protein being studied do not accidently get removed by the action of protein phosphatases that do not dephosphorylate this protein in the actual physiological scenario.

Maybe the phosphorylated protein and these phosphatases exist in different cellular compartments and cannot reach each other in the native biological scenario. However, during cell lysis for protein extraction these phosphatases somehow get in contact with this phosphorylated protein, and dephosphorylates it.

If this happens, it may lead to experimental artifacts – faulty data due to technical errors.

A way around this is using phosphatase inhibitors in your experiment, as we will discuss in the next section.

What are phosphatase inhibitor cocktails?

A phosphatase inhibitor cocktail contains multiple compounds that inhibit the different types of protein phosphatases.

Like a cocktail drink made of many different types of spirit beverages, a phosphatase inhibitor cocktail contains an assortment of different inhibitor compounds that block the function of the variety of protein phosphatases found in biological systems.

GoldBio sells three different types of phosphatase inhibitor cocktails, targeted against a wide range of phosphatases.

Why are phosphatase inhibitor cocktails used in experiments?

Compounds in phosphatase inhibitor cocktails block phosphate removal by different protein phosphatases. They are used in protein extraction and purification to preserve the phosphorylation status of the target proteins just as they exist in the physiological environment they are being studied.

Here is an explanation with the help of a schematic figure.

Figure 5. Illustrates how phosphorylated target proteins would potentially become exposed to phosphatases during cell lysis; however, phosphatase inhibitors and cocktails protect target proteins from accidental dephosphorylation during cell lysis.

As shown in figure 5, the phosphorylated target proteins might be shielded from cellular phosphatases by some kind of physiological means in the actual biological setup, like inside the cell. This prevents them from being dephosphorylated. This is depicted in panel A of figure 5.

As a researcher, you want to study the target proteins exactly as they exist within the cell at a given physiological time point – in this case, in phosphorylated forms.

To put things in perspective, think about phosphorylated Tau proteins from Alzheimer’s brain tissue.

During cell lysis for extraction of your phosphorylated target protein, the cell compartments get broken. And this phosphorylated protein comes in contact with some protein phosphatases. These enzymes dephosphorylate the target protein in your sample. This is what you see in panel B of figure 5.

Because of this, ultimately when you study the extracted target protein, you find it without the phosphorylation; just opposite to how it exists in the physiological context you are studying the target protein in.

So, as you see, these are wrong results that your experiment yields because of a technical error in the experiment – that the phosphatases contaminated your target protein.

But, as an experienced researcher, in the protein extraction steps, you use high quality phosphatase inhibitor cocktails.

How this is helpful is shown in panel C of figure 5.

Compounds in the phosphatase inhibitor cocktail block the various different phosphatases from dephosphorylating your target protein. And you are able to extract or purify your target protein in its phosphorylated form, exactly how it exists in the physiological scenario being studied.

If you read our article on protease inhibitor cocktail, phosphatase inhibitor cocktails are useful in pretty similar lines – just that while protease inhibitors prevent proteases from degrading your target proteins, phosphatase inhibitor cocktails block phosphatases from dephosphorylating your target proteins.

References

• Liu et al. 2005. Dephosphorylation of tau by protein phosphatase 5: impairment in Alzheimer's disease. J Biol Chem
• Leijten et al. 2022. Histidine phosphorylation in human cells; a needle or phantom in the haystack? Nature methods. 19, 827-828
• Taylor and Dixon. 2000. Myotubularin, a protein tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates the lipid second messenger, phosphatidylinositol 3-phosphate. PNAS. 97(16): 8910-5
• Tonks 2006. Protein tyrosine phosphatases: from genes, to function, to disease. Nature reviews molecular cell biology. 7, 833-846
• Shi 2009. Serine/Threonine Phosphatases: Mechanism through Structure. Cell. 139 (3):468-484
• Schetinger et al. 2007. NTPDase and 5'-nucleotidase activities in physiological and disease conditions: new perspectives for human health. Biofactors. 31(2):77-98
• Bogan and Brenner. 2010. 5′-Nucleotidases and their new roles in NAD⁺ and phosphate metabolism. New journal of chemistry
• Rajas et al. 2019. Glucose-6 Phosphate, a Central Hub for Liver Carbohydrate Metabolism. Metabolites. 9(12):282
• Fernandez-Sanchez et al. 2013. Comparative Toxicological Study of the Novel Protein Phosphatase Inhibitor 19-Epi-Okadaic Acid in Primary Cultures of Rat Cerebellar Cells. Toxicological sciences. 132(2):409-418