Protein Phosphatase: Overview & Applications

Protein Phosphatase: Overview & Applications

Protein Phosphatase: Overview

Protein phosphatase is a family of enzymes that remove phosphate groups from proteins. The reversible phosphorylation (through protein kinase enzyme) and dephosphorylation processes control the metabolic process of organisms, such as cell signaling, gene expressions, cell cycle progression, ion transport, and control protein function. 

The phosphate groups are mainly attached to amino acid residues, such as threonine, tyrosine, and serine residues, by the kinase enzyme and removed by phosphatases. Based on their structure, substrate specificity, and activity on a specific amino acid group, the phosphatase enzyme is divided into the following families

  • Protein serine/threonine phosphatase (PSPs): It’s also known as ser/thr phosphatase, which causes dephosphorylation of phosphoserine/threonine residues. The phosphatase is further divided into sub-families of metal-dependent protein phosphatases (PPMs), phosphoprotein phosphatases (PPPs), and aspartate-based phosphatases. 

The phosphoprotein phosphatases consist of enzymes, such as protein phosphatase 1 (PP1), protein phosphatase 2a (PP2A), and PP4, PP5, PP6, PP7, and calcium-activated PP2B (or calcineurin).

The metal-dependent protein phosphatases (PPMs) sub-family includes enzymes, such as pyruvate dehydrogenase and PP2C. An example of aspartate-based phosphatase includes transcription initiation factor II, such as TFIIF-associated C-terminal domain phosphatase. 

  • Phosphotyrosine phosphatase (PTPs): The enzyme removes the phosphate group from tyrosine residues. It’s mainly involved in signal transduction pathways, such as the AKT signaling pathway and MAP kinase pathway. Some examples of protein tyrosine phosphatases include dual specificity phosphatase (phosphatase and tensin homolog protein (PTEN)), MAP kinase phosphatases (MKPs), and T cell receptor (TCR) signaling. 

Figure: Classification of protein phosphatases, based on their activity and localization, such as cytosolic or transmembrane.

In this article, we will cover further the structure of the phosphatase enzyme, its function in organisms, and its working mechanisms.

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How Does Protein Phosphatase Work?

Protein phosphorylations and dephosphorylation mechanisms strongly regulate cellular responses in organisms in response to external stimuli. The activity of the phosphatase enzyme, which causes the dephosphorylation of proteins, is regulated by its three domains:

  • Regulatory domain: It’s involved in the localization of proteins to a subcellular compartment and regulates their interaction with other molecules. The regulatory subunits modulate proteins’ functions by modifying them through allosteric modifications using reversible protein phosphorylation and second messengers. They also contain binding sites for regulatory molecules, such as Ca2+, that through concentration change control the enzyme’s activation or inhibition during the process. 
  • Target domains: They are involved in the interaction of enzymes with specific proteins. For example, the PP5 phosphatase has three tetratricopeptide repeat (TPR) domains for protein-protein interaction in vivo. 
  • Catalytic domain: The domain is highly conserved among different phosphatases. For example, in PP1, PP2A (the amino acid sequence consists of 37kDa), and PP2B phosphatase enzymes the catalytic subunit is composed of 280 residues. The enzymes are mainly identified by their distinct regulatory subunits. 

Protein Phosphatase Working Mechanism

Dephosphorylation is the process of detaching phosphates as free ions into a solution. This process does not require energy in the form of ATP like the phosphorylation reaction. Each phosphatase follows different mechanisms to act on its target substrates. 

For example, the cysteine-dependent phosphatase initiates the process through a nucleophilic attack on the phosphate group of the target protein. This is followed by the protonation of tyrosine residue, formation of phospho-cysteine intermediate, and release of phosphate ion through hydrolysis which regenerates the active phosphatase. 

The Metallo-phosphatases start the dephosphorylation process by attacking the target protein through its hydroxyl group that bridges two metal ions present in the active site of the enzyme. The nucleophilic attack breaks the bond between the substrate and phosphate group, completing the dephosphorylation reaction. The active site of the enzyme is then freed through a hydrolysis reaction, and the enzyme is regenerated for further dephosphorylation reactions. 

What is Protein Phosphatase Used For?

Protein phosphatase regulates many biological processes opposite to protein kinase. The balance of both enzymes is important for the modulation of the cell cycle progression (such as through Ca2+/Calmodulin-dependent signaling pathways), signal transduction pathways, ion transport, transcription, translation, mitosis, and motility processes.

Figure: Functions of protein phosphatase.

Cell Regulation

Phosphatase enzymes regulate cellular processes by modulating the activity and functions of proteins involved in the processes, such as gene expression, signal transduction, apoptosis, cell growth and differentiation, and cell cycle progression. 

For example, dephosphorylation of NDEL1 at CDK1 sites by the PP4 enzyme facilitates its binding to LIS1-Dynein. This causes the pulling force by microtubules on spindles for symmetric cell division. 

Figure: Role of PP2A in cell cycle progression. 

Phosphate Removal

Phosphatase enzymes have a crucial role in regulating cellular functions and processes, such as cell division and signal transduction. Different isoforms of phosphatase enzymes have their unique mechanisms for detecting target proteins and removing phosphate groups. However, most of them start the dephosphorylation reaction through nucleophilic attack on the phosphate group of the target substrate, followed by hydrolysis of the phosphate ions to regenerate the enzyme. 

Phosphatase enzyme is also involved in regulating blood glucose levels by dephosphorylating enzymes, such as glycogen phosphorylase, phosphorylase kinase, and glycogen synthase. The process causes the activation of the enzyme glycogen synthase and the deactivation of glycogen phosphorylase and phosphorylase kinase enzymes that lower sugar levels. 

What Industries Use Protein Phosphatase?

Protein phosphatase enzyme has crucial roles in regulating in vivo functions and activities of proteins. It makes the enzyme even more important in molecular biology and biochemistry labs for the study of biological processes and signaling pathways in vitro.


In biotech labs, researchers isolate and purify the phosphate enzymes using recombinant techniques, cloning approaches, combined proteomic and genomic approaches, and different biochemical assays for various research studies, such as cell signaling and cell cycle regulation studies. 

The effect of the enzyme in a process is determined using high-throughput methods such as biochemical assay, mass spectrometry, and chromatography. Additionally, the use of phosphatase inhibitors, such as okadaic acid, to block phosphatase functions helps researchers understand the phosphatase activity in a process.

Medical Research

Phosphatases play a pivotal role in regulating signal transduction pathways within organisms, including the PI3K/Akt/mTOR (Mammalian target of rapamycin)-dependent signaling cascade. For example, cancer cells often exhibit elevated Akt activity, a condition that can be mitigated through dephosphorylation mediated by the enzyme Protein Phosphatase 2A.

Likewise, phosphatase PTEN assumes a crucial role in modulating the PI3K signaling pathway while also contributing to cell cycle arrest and DNA repair. Given their indispensable functions in organisms, researchers are dedicated to unraveling the mechanisms through which phosphatases operate in conjunction with pathways associated with severe ailments like cancer. 

The ultimate goal is to develop effective treatments that target the degradation of proteins responsible for triggering these diseases. 

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Protein phosphatase is an enzyme family responsible for the dephosphorylation of proteins. Alongside protein kinase enzymes, they play a vital role in regulating cellular functions, including cell cycle progression, apoptosis, and signal transduction.

Several isoforms of the enzyme exist, which can be classified into two main families: Protein serine/threonine phosphatase (PSPs) and Phosphotyrosine phosphatase (PTPs). Each enzyme possesses its unique structure and functional characteristics. 

The phosphatase enzymes are crucial components of in vitro studies aimed at understanding signaling pathways, particularly those associated with life-threatening diseases like cancer. Conducting such high-throughput studies often demands advanced equipment and high-quality reagents, which can be challenging for labs with limited funding.

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