28.6: Phosphatases

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Learning Goals (ChatGPT o3-mini)

Below is a series of learning goals to help you master the mechanisms, structural features, and regulatory roles of protein phosphatases in intracellular signaling:

1. Fundamental Concepts of Reversible Phosphorylation:

   • Explain how protein phosphorylation and dephosphorylation regulate protein function and signaling pathways.
   • Describe how kinases and phosphatases work in concert to modulate cellular responses.

2. Classification and Structural Characteristics of Phosphatases:

   • Differentiate among the main families of phosphatases, including:
     • Serine/Threonine Phosphatases: Understand the roles of PP1, PP2A, PP2B (calcineurin), and PP2C (PPM family), focusing on their metal dependency and active site architecture.
     • Protein Tyrosine Phosphatases (PTPs): Outline the structural features that distinguish PTPs (e.g., the active site Cys in a CX₅R motif, WPD loop) and their mechanism of catalysis.
     • Dual Specificity Phosphatases: Describe how proteins like PTEN function as both protein and lipid phosphatases and regulate key signaling pathways.

3. Regulatory Subunits and Targeting Specificity:

   • Discuss how phosphatases gain substrate specificity through the binding of regulatory subunits (e.g., the glycogen-targeting subunits for PP1) and how these interactions influence localization.
   • Explain the role of short linear motifs (SLiMs) and domains (e.g., SH2 domains in PTP regulatory complexes) in substrate recognition.

4. Catalytic Mechanisms and Active Site Chemistry:

   • Describe the role of divalent metal ions (such as Mn²⁺, Mg²⁺) in the catalytic mechanism of phosphatases, including their function in lowering the pKa of water to generate a nucleophilic hydroxide.
   • Compare and contrast the active site features of Ser/Thr phosphatases (e.g., binuclear metal clusters in PP1/PP2A) with those of PTPs (e.g., the active site cysteine and the WPD loop).

5. Structural Insights Through Visualization:

   • Analyze interactive structural models (e.g., iCn3D) to identify key catalytic residues and substrate-binding sites in phosphatases such as PP1, PP2A, and PTP1B.
   • Explain how inhibitors (e.g., microcystin for PP1, okadaic acid for PP2A) bind to the active sites and what these interactions reveal about enzyme function.

6. Regulation of Metabolic and Signaling Pathways:

   • Explain how phosphatases like PP1 regulate glycogen metabolism by activating glycogen synthase and inhibiting glycogen phosphorylase via dephosphorylation.
   • Describe how dual specificity phosphatases such as PTEN regulate lipid signaling (e.g., PI3K-AKT pathway) and act as tumor suppressors.

7. Clinical Relevance and Drug Targeting:

   • Evaluate the significance of phosphatase dysregulation in diseases such as cancer and metabolic disorders.
   • Discuss strategies for designing selective phosphatase inhibitors and the challenges involved due to the conserved nature of active sites in many phosphatases.

These learning goals will guide you through understanding how protein phosphatases function at the molecular level, how their activities are regulated and targeted, and why these processes are critical for proper cellular signaling and overall cell function.

Introduction

Protein kinases phosphorylate proteins in a process that can either activate or inhibit the target protein's activities. To control signaling processes, the activities altered by protein phosphorylation can be readily reversed by the dephosphorylation of Ser-, Thr-, or Tyr-phosphoesters through simple hydrolysis. Protein phosphatases catalyze these reactions. Some of these phosphatases also cleave phosphates from lipids.

There are three main families of phosphatases: phospho-Tyr phosphatases (PTP), phospho-Ser/Thr phosphatases, and those that cleave both. Of all phosphorylation sites, most (86%) are on serine, 12% involve threonine, and about 2% are on tyrosine. Molecular size, inhibitors, divalent cation requirements, and other factors can also be used to categorize them. In contrast to kinases, which differ in the structure of their catalytic domains, many protein phosphatases (PPs, also abbreviated Ppp for Protein phosphatases) gain specificity by binding protein cofactors that facilitate translocation and binding to specific phosphoproteins. The active phosphatase, hence, often consists of a complex of the phosphatase catalytic subunit and a regulatory subunit. Regulatory subunits for Tyr phosphatases may contain a SH2 domain, allowing binding of the binary complex to autophosphorylated membrane receptor Tyr kinases.

We'll consider examples of all four families. They recognize target proteins through protein-protein interactions and specific binding site motifs. There are over 10,000 pSer and p-Thr sites on target proteins, so targeting specific sites must be quite nuanced.

Serine/Threonine Phosphatases

Important Ser/Thr phosphatases (PPs for Protein Phosphatases) include:

• Protein phosphatase 1 (PP-1 or Ppp1) - This is the most abundant PPP in humans. Different regulatory subunits target this to the liver glycogen particles (GL subunit), striated muscle glycogen, and sarcoplasmic reticulum (GM subunit) or smooth muscle fibers (M subunit). It is also present in the nucleus, where it is presumably involved in the regulation of transcription factors. It is also involved in RNA splicing and signaling at neural synapses.
• Protein phosphatase 2A (PP-2A or Ppp2) - is a trimer with catalytic, regulatory, and scaffolding (also regulatory) structural subunits. It is primarily located in the cytoplasm and is involved in numerous cellular processes.
• Protein phosphatase 2B (PP-2B or Ppp3) - also called calcineurin or Ca²⁺/Calmodulin dependent protein phosphatase - It consists of a catalytic subunit (calcineurin A) and a regulatory, calcium-binding subunit (calcineurin B). It is inhibited by the complex of the immunosuppressant cyclosporin and FK506 with immunophilins. PP2B regulates PKA and PKC
• (PP2C) -  are Mg²⁺/Mn²⁺- dependent serine/threonine phosphatases that are involved in cell growth, cellular stress responses, and signaling pathways in the brain and are also important in plants for drought, cold, and salt stress. 

The catalytic subunits of PP1, 2A, and 2B share a great deal of amino acid homology, and based on this homology, belong to one family. PP2C belongs to another family. PPs are often categorized into three other families, including phosphoprotein phosphatases (PPPs) and metal-dependent protein phosphatases (PPMs). There are about 30 catalytic PP subunits (many fold fewer than Ser/Thr Kinases). They gain specificity by binding numerous modulatory regulatory subunits.

As with other proteins, the names assigned to proteins when they were discovered often do not reflect an organizational scheme that would name different members based on structural similarities. PP-1, 2A, and 2B are better named Ppp1, Ppp2, and Ppp3 which denote members of the Protein PP (PPP) family. PP-2C would be named Ppm1 as the first member of the PPN family. All PPPs have three short sequence motifs that bind divalent cations.

Additionally, older names for the given PPs referred to both the catalytic subunit and the dimer, which includes the regulatory subunits. For clarity, the name of the catalytic protein phosphatase 1 is PP1c, and its regulatory subunits are referred to as RIPPOs, which are regulatory interactors of protein phosphatase one.

Protein Phosphate-1 (PP-1):

PP-1 is involved in numerous signaling pathways that regulate cell division, protein synthesis, and other cellular processes. It catalyzes most serine–threonine dephosphorylation in cells. It is perhaps best known for its regulation of glycogen mobilization. Insulin signals the well-fed state in healthy people and promotes glucose uptake through the GPCR insulin receptor, which we discussed in Chapter 12.4. Under these conditions, excess glucose is used to elongate glycogen, our main carbohydrate energy storage polymer.

In contrast, the starving or low-energy state is signaled by the hormone glucagon. You would expect that signaling pathways activated in the presence of insulin would promote glycogen synthesis and inhibit glycogen breakdown. PP-1 is a key factor in the regulation of both processes:

• Insulin activation of glycogen synthesis - PP1 dephosphorylates glycogen synthase (the enzyme that synthesizes glycogen) and in the process ACTIVATES it.
• Insulin inhibition of glycogen breakdown - PPI dephosphorylates two key enzymes involved in glycogen breakdown, phosphorylase kinase and glycogen phosphorylase a (with a pSer14), and in the process, INHIBITS them.

PP1c interacts with many different regulatory subunits (RIPPOs) forming unique heterodimers. The regulatory subunits also bind potential substrates for PP1c and help localize the enzyme at specific sites. The regulatory subunit involved in the PP1c effect on glycogen is called the glycogen-targeting subunit, G_M. There are seven such regulatory subunits involved in the metabolism of glycogen. GM (RGL) is expressed in muscles, and GL is expressed in the liver. All of the regulatory subunits (unfortunately referred to as G-subunits) have a conserved RVxF amino acid sequence that interacts with specific sites on the catalytic subunit, typically located distal to the active phosphatase site. Binding through the RVxF sequence does not affect the active site of the PP-1c. The binding of the regulatory subunit to the PP-1c can also occur outside of the canonical RVxF sequence. The regulatory subunits also contain a starch-binding domain (SBD), also known as the carbohydrate-binding module (CBM21). The subunits are often highly disordered until they are bound.

Figure \(\PageIndex{1}\) shows an interactive iCn3D model of protein phosphatase 1 (PP1) bound to the muscle glycogen-targeting subunit (Gm) and microcystin (6DNO) and the toxin microcystin.

Figure \(\PageIndex{1}\): Protein phosphatase 1 (PP1) bound to the muscle glycogen-targeting subunit (Gm) and microcystin (6DNO). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...6f8f2kBRW4uhv5

PP-1 alpha catalytic chain is shown in gray. Its active site, where it binds Ser/Thr phosphorylated proteins, is shown in magenta. Bound in that site is the toxin microcystin. The small chain shown in cyan is protein phosphatase 1 regulatory subunit 3A. PP-1 binds to its regulatory sequence, a 65RVxF68 sequence common to many regulatory PP-1 subunits. This subunit contains a serine 67 that is phosphorylated by protein kinase A. This inhibits the binding of the catalytic and regulatory subunits. (PKA) of the “x” residue in the G_M RVxF motif, Ser⁶⁷_GM, inhibits PP1 binding (16). Val79 and Lys80 on GM also form a motif that binds a corresponding pocket in the catalytic subunit.

Note the pi-cation (red) and pi-stack (blue) interactions from Phe 68 of the G_M 65RVSF68 motif of the regulatory subunit to the PPI catalytic subunit. Microcystin is a 7-mer peptide ring that has five noncanonical amino acids and two regular ones. A covalent bond from Cys273 of PP1 (labeled in the above iCn3D model) to the methyl-dehydroalanine (Mdha) of the toxin forms. Toxic cyanobacteria produce microcystins, which are highly toxic and lethal, especially to animals, including humans, that drink water contaminated with these cyanobacteria. They will pose a greater threat in a warming world from climate change. They also bind to PP-2A.

Another example of a regulatory subunit for PP1 is the PP1 nuclear target subunit (PNUTS). The activity of the complex in the nucleus regulates the phosphorylation state o many proteins involved in the cell cycle including p53, a tumor suppressor in many tumors. It also regulated chromatin structure and RNA processing. As with the muscle glycogen-targeting subunit (Gm), PNUTS is intrinsically disordered when not bound to PP1 and is very extended when bound. The catalytic subunits of PP1c and PP2A have an acidic, hydrophobic, and C-terminal groove. PNUTS binds one of the substrate grooves at an arginine subsite, but not the active site.

So far, we can say that the specificity of the PPP binding is determined to some degree by the regulatory subunits. We turn to the contributions of the catalytic specificity of PP-1c when we compare it to PP-2Ac below.

Mechanism of PP1-A and PP-2A and B

The catalytic subunits of PP-1 and PP-2 are very homologous, with nearly identical key active site side chains. The site contains two Mn²⁺ ions very close to each other, as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Interactions with two Mn ions in the active site of protein phosphatase-1 (PP-1). After McWirter et al. J. Am. Chem. Soc. 2008, 130, 41, 13673–13682

The figure crudely shows that each Mn²⁺ is octahedrally coordinated to side chains and one oxygen from a water molecule for each metal ion. Water 2 (or more likely OH^-) and an aspartate D92 oxygen bridge the two ions. The phosphate of the target Ser-OPO₃^2- or Thr-OPO₃^2-, or an inhibitor such as tungstate, also bridges the metal ion.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of Human Protein Phosphatase 1 Active Site Residue (4mov) which shows the same active site residues

Figure \(\PageIndex{3}\): Human Protein Phosphatase 1 Active Site Residue (4mov). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?MYPrWfkX1PBaAggL9

The metals, singly or in combination, probably reduce the pKa of bound water to produce the deprotonated hydroxide, which then engages in an S_N2 attack on the phosphate. Hence, the metal ions act as an electrostatic catalyst.

The subtle differences in the active site and the three groups contribute to the specificity of the PPPs.

Comparison of the catalytic subunits of PP1A and PP2A

As mentioned above, substrate specificity is altered by subtle changes in the active site and the grooves of PPPs. Figure \(\PageIndex{4}\) shows the acidic groves for PP1c and PP2Ac.

Figure \(\PageIndex{4}\): Acidic groves for PP1c (3EGG, chain A) and PP2Ac (4I5L, chain C)

The color is the electrostatic surface potential, with red indicating negative and blue (notably absent) positive. The acidic groove is stronger in PP1c. The asterixis * shows the catalytic cleft containing two Mn²⁺ ions. Hoermann et al. Nature Communication | (2020) 11:3583 | https://doi.org/10.1038/s41467-020-17334-x. Creative Commons Attribution. 4.0 International License, http://creativecommons.org/licenses/by/4.0/.

The negative acidic groove is highly enriched in negatively charged side chains. Figure \(\PageIndex{5}\) shows the actual amino acids contributing to the negative electrostatic potential in aligned PP1c and PP2Ac.

Figure \(\PageIndex{5}\): Amino acids contributing to the negative electrostatic potential in aligned PP1c and PP2Ac. Zhang et al. Hoermann et al. ibid

The orange spheres show the location of acidic side chains. PP1c is shown in blue/black and PP2Ac in red/gray.

The stronger acidic groove in PP1Ac gives it a greater preference for pSer/pThr-protein targets with basic motifs than PP2Ac, in a manner that is independent of the bound regulator subunit. In contrast, PP2A requires interaction with regulatory subunits of a more acidic composition to target basic motifs in protein targets. These features for PP1c and PP2Ac are compared in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): Comparative activity of PP1 and PP2A to basic target protein substrates.

Panel (d) shows that the holoenzyme for PP1 has a greater preference for positive basic motifs than the holoenzyme for PP2A, which needs to associate with a negatively charged regulatory subunit for activity towards target proteins with basic motifs.

Panel (e) also shows that the catalytic subunits of both PP1 and PP2A prefer p-Thr protein targets compared to p-Ser.  Velocity versus substrate graphs also show these effects.  Both PP1 and PP2A holoenzymes have a preference for pT due to the higher catalytic efficiency of their respective catalytic subunits towards pT over pS. Hoermann et al. ibid.

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the protein phosphatase 2A catalytic subunit in complex with a larger regulatory subunit and bound to the phosphatase inhibitor and tumor promoter okadaic acid (2IE4). The toxin, found in sponges and shellfish, is produced by dinoflagellates.

Figure \(\PageIndex{7}\): Protein phosphatase 2A core enzyme (catalytic and regulatory subunit) bound to okadaic acid (2IE4). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Sb2Vq5iZgbBSz8

The regulatory subunit is depicted in a secondary structure color scheme. This scaffolding protein is shaped like a horseshoe. The phosphatase inhibitor okadaic acid is shown in spacefill bound to the catalytic subunit, shown in gray. The side chains of the catalytic subunit interacting with the 2 Mn²⁺ ions are shown in CPK-colored sticks (zoom in to see them). Upon binding the catalytic subunit, the scaffolding regulatory subunit is quite flexible and adaptable in its interactions with other proteins.

Protein Phosphate 2B: Calcineurin (CN)

Calcineurin (CN), or PP2B, is depended on Ca²⁺. It is involved in the development, immune signaling, and heart function. It consists of a catalytic site (CNA) and a calcium-binding regulatory subunit, CNB, so it is another example of PPP heterodimers. CNA has a catalytic domain and domains that bind CNB (the regulatory subunit), calmodulin (CAM, a calcium-binding protein that we will explore more in the next chapter section), and an autoinhibitory domain that blocks the active site. On Ca²⁺ release from internal organelles, the ion binds to both CNB and CAM. These events cause conformational changes that release the bound autoinhibitor.

As with the other phosphatases, much effort has been made to determine how CN interacts with specific pSer- and p-Thr sites on targets. We'll focus on one, the integral membrane Na⁺/H exchanger 1 (NHE1). This protein is itself regulated by Ca²⁺ ions and by phosphorylation by kinases we have previously studied, the MAPK ERK1/2 and the JNK kinase. Erk2 phosphorylates NHE1 at 6 Ser/Thr side chains in the recognition sequences. Several different phosphatases, including CN, can regulate NHE1 activity through direct dephosphorylation.

CN binds short linear motifs (SLiMs) named PxIxIT and the LxVP that are found in interacting partners, including regulatory subunits, as well as inhibitors and substrates. As we saw above, the regulatory subunits of PP1A and PP2A are highly disordered. Likewise, SLiMs are on intrinsically disordered regions as well as interacting proteins.

• PxIxIT binds to the catalytic domain of CNA22. It also enables interaction between CN and NHE1.
• LxVP binds to a cleft between the CNA and CNB, which is only available in the active form of the protein.

CN doesn't dephosphorylate multiple nearby p-Ser side chains of NHE1 (pS363, pS723, and pS726) since they are close to the NHE1-PxIxIT interaction sit,e which sterically restricts their binding to the active site. However, the three other phospho S/T sites on NHE1 (pS771, pS785, and the actual target site, pT779) are sufficiently distant from NHE1's PxIxIT site to interact with the CN active site. The T779S mutation demonstrates that dephosphorylation of the phosphorylated version occurs at a faster rate for pT779 and a slower, yet reasonable rate for pS779. Therefore, other specificity factors are in play. A newly discovered very short 4-amino acid site motif in NHE1, including pS779, appears to be a source of selectivity. This TxxP motif in NHE1 is 779TPAP782. Such short recognition motifs differ from the selection of substrates by PP1, which involves multiple domain binding interactions and steric restrictions imposed by them.

Figure \(\PageIndex{8}\) shows a model of the structure of the NHE1 exchanger (left panel a) and the calcineurin CNA/CNB complex.

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Figure \(\PageIndex{8}\): Docking motifs mediate the interaction of NHE1ct with CN

The motifs present in NHE1 (LxVP and PxIxIT) in the intrinsically disordered tail of NHE are indicated in the left panel, and their corresponding binding sites in the CNA dimer are shown in corresponding colors. The calmodulin binding site is also shown. Erk2 phosphorylation sites in NHE1 are shown in panel C, along with the consensus motif sequences (PxIxIT in purple, LxVP in orange, and TRAP (uncolored)). Hendus Altenburger et al. Nature Communication (2019) 10:3489 | https://doi.org/10.1038/s41467-019-11391-7Creative Commons Attribution 4.0 International License. http://creativecommons.org/ licenses/by/4.0/.

Figure \(\PageIndex{9}\) shows an interactive iCn3D model of calcineurin (PP2B) complex bound to a peptide from the Na⁺ /H⁺ -exchanger 1 (6NUC)

Figure \(\PageIndex{9}\): calcineurin (PP2B) complex bound to a peptide from the Na⁺ /H⁺ -exchanger 1 (6NUC). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...GgqSWpirpnZGt9

Protein Phosphatase 2C

The protein phosphatase 2C (PP2C) is a member of a family of metal-dependent protein phosphatases sometimes abbreviated as PPMs. (Of course, the other phosphatases we discussed above are also metal-dependent.) The required Mg²⁺ or Mn²⁺. PP2C is a monomeric enzyme with at least four isoforms in humans. In humans, there are at least 17 members. One is unfortunately named protein phosphatase 1D, but also referred to as PPM1D, PP2Cδ, or Wip1. It is involved in heterochromatin silencing and the cycle. Mutations in its gene can accordingly lead to the development of tumors.

PP2Cs in humans have a binuclear metal cluster which reduces the pKa of water, producing OH^- for S_N2 attack on the phosphorus in the pSer or pThr in target phosphorylated proteins. Figure \(\PageIndex{10}\) describes binding interactions around the binuclear site in PP2Cα.

Figure \(\PageIndex{10}\): Binding interactions around the binuclear site in PP2Cα. after Pan et al. Sci Rep 5, 8560 (2015). https://doi.org/10.1038/srep08560htt...cles/srep08560

The metal binding site includes several water molecules. Interactions with the metals are very different than the PP1 and PP2 active site shown in Figure \(\PageIndex{2}\).

If Cd is bound at the M1 site, the activity of the enzyme is blocked, so it is required for catalysis. Making the mutations affecting M2 (D38A and D38K) suggests that M2 is involved in binding the phosphate of the substrate, and also stabilizes the transition state and the leaving group in the reaction. H62 probably acts as a general acid catalyst.

Not all PPCs require both metal ions. The plant hormone abscisic acid regulates stress responses in plants. When it binds to a particular receptor called PYL1 (alternative name PYR1-like protein 1), the receptor interacts with a PP-2C called ABI1 (also called Abscisic acid-insensitive 1). Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the ternary complex of Abscisic acid, PYL1 and ABI1 (phospholipase 2C) (3KDJ)

Figure \(\PageIndex{11}\): Ternary complex of Abscisic acid, PYL1, and ABI1 (phospholipase 2C) (3KDJ Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?FbEeZ3n5dWvA6bp56

The catalytic subunit, which, in contrast to PP1, PP2A, and PP2B, contains only one Mn²⁺ ion, is depicted in gray, with amino acid side chains interacting with the metal ion labeled. The cyan subunit serves as the receptor for abscisic acid, as illustrated in the spacefill model.

Protein Tyrosine Phosphatases

Protein Tyr phosphatases (PTPs) consist of receptor-like (transmembrane) and intracellular Tyr phosphatases. They more resemble tyrosine kinases in their complexity than the Ser/Thr phosphatases. There are about 100 PTPs in the genome, a number similar to the number of protein tyrosine kinases. PTPs have an active site Cys in a CX5R-(S/T) motif with an active site Cys nucleophile and an Arg in the phosphate binding (P) loop. Some examples we will discuss include:

• Low-molecular-weight PTPase - These enzymes play roles in cellular metabolism and differentiation. They have a molecular weight of 18,000 and have an active site CX5R-(S/T) motif, where the C (Cys) is an active site nucleophile.
• PTP1B - dephosphorylates many cell surface receptors (insulin, EGF, PDGF) that have been phosphorylated on Tyr residues. Its main activity appears to be dephosphorylating nascent receptors in the endoplasmic reticulum before they reach their final destination at the cell membrane.
• Tyrosine phosphatase nonreceptor type 11, ptpn11, commonly called SHP2

Figure \(\PageIndex{12}\) shows the protein tyrosine phosphatase (PTP) superfamily.

Figure \(\PageIndex{12}\): Protein Tyrosine Phosphatase superfamily Abdelsalam et al. Biomolecules 2019, 9, 286. https://doi.org/10.3390/biom9070286. Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0/.

In contrast to the active site of the Ser/Thr phosphatases like PP-1, PP-2A, and PP-2B, the active sites of protein tyrosine phosphatases (PTPs) do not have a bimetal ion cluster in the active site. Instead, they all have an active site cysteine that acts as a nucleophilic catalyst in the hydrolysis of the p-Try phosphoester bond. The active site PTP domain is found in all proteins, so they all use similar catalytic mechanisms, as shown in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): General catalytic mechanism of protein tyrosine phosphatases

The phosphotyrosine side chain of the target phosphoprotein binds in the phosphate-binding P-loop (H/VCxxxxxRS/T), which contains the nucleophilic Cys 12 and Arg 18 that stabilize the charge on the phosphate. The Asp in the WPD loop positioned across from the nucleophilic Cys 12 acts as a general acid. Since the active site is nearly identical across PTPs, it has been challenging to design drugs that bind to the active site while also being selective for specific PTPs.

Low molecular weight protein tyrosine phosphatase - LMW-PTP

This protein tyrosine phosphatase is the simplest in structure among all protein tyrosine phosphatases. It features the phosphate-binding P-loop (12CxxxxxR18), comprising the nucleophilic Cys 12 and Arg 18, which stabilizes the charge on the phosphate. It does not have the conserved WPD loop but utilizes Asp 129, positioned across from Cys 12, as a general acid. This enzyme exists as two main isozymes, A and B. Figure \(\PageIndex{14}\) shows an interactive iCn3D model of human low molecular weight protein tyrosine phosphatase bound to sulfate (1xww).

Figure \(\PageIndex{14}\): human low molecular weight protein tyrosine phosphatase bound to sulfate (1xww). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...jaW2syCWWeeLD8

The active site is deep as shown in Figure \(\PageIndex{15}\).

Figure \(\PageIndex{15}\): Active site pocket and surround surface of low molecular weight protein tyrosine phosphatase (1xww)

The sulfate, a mimetic for the phosphate on the p-Try protein target, is deeply buried. The CPK-colored surface (green, red, blue) around the sulfate is the side chains of Tyrosine 131 and 132, as well as Trp 49. Y131 and Y132 are part of a loop containing Asp 129, the general acid. This loop is analogous to the WPD loop. The three aromatic amino acids on top of the pocket make it deep enough that pSer and pThr side chains can't reach the active site nucleophile Cys 12. Trp 49 is also in a variable loop (34 amino acids, shown as an orange surface) that differentiates two of the major isozymes, the A and B forms, and contributes to substrate binding specificity.

Tyrosine-protein phosphatase non-receptor type 1, also known as PTPN1 or PTP1B

PTP-1B regulates the endoplasmic reticulum unfolded protein response and is involved in insulin JAK/STAT and HER2 (ErbB2) signaling. It has a full-length form (MW 50,000) and a C-terminal shortened form (37,000)

Given the difficulty in targeting the active site, which is common to all PTPs, efforts have concentrated on the development of allosteric inhibitors that bind to exosites located away from the active site. An example is trodusquemine (MSI-1436) used in the treatment of obesity and type 2 diabetes. It binds much more tightly to the full-length form.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of the human Protein Tyrosine Phosphatase 1B (1-301) in complex with the inhibitor OTA (5K9W).

Figure \(\PageIndex{16}\): Human Protein Tyrosine Phosphatase 1B (1-301) in complex with the inhibitor OTA (5K9W). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...fTurfTTFiW2b47

The P-loop is shown in magenta, the WPD loop in cyan, and the substrate binding loop (SBL), which allows entry of pTyr but not pSer and pThr, in blue. The key side chain in the P-loop (Cys 215 and Arg 221) as well as the catalytic general acid (Asp 181) are shown in sticks and labeled. The inhibitor is shown in spacefill and CPK colors. The movement of the WPD loop is rate-limiting for the hydrolysis of P-Tyr esters. On binding, the WPD starts to close, and in the process, Arg 221 moves to form salt bridges with the phosphate. The complete closure of the WPD then follows, positioning Asp 181 for general acid catalysis. Key interactions of PTP-1B phosphoprotein in insulin and cytokine signaling are shown in Figure \(\PageIndex{17}\).:

Figure \(\PageIndex{17}\): Protein tyrosine phosphatase 1B (PTP1B) and its effects on signaling. Maja Köhn ACS Cent. Sci. 2020, 6, 4, 467–477. Publication Date: March 13, 2020. https://doi.org/10.1021/acscentsci.9b00909. This is an open-access article published under an ACS Author Choice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Panel (A, left) shows how PTP1B dephosphorylates the insulin receptor and the insulin receptor substrate (IRS), which we have explored in a previous section. Panel (A, right) illustrates its activity in the JAK/STAT pathway, which we have previously observed. One cytokine receptor that it regulates is the leptin receptor. The hormone leptin, released from adipocytes, plays a key role in regulating lipid metabolism. Panel B shows the structures of key inhibitors of PTP-1B.

Protein tyrosine phosphatase nonreceptor type 11 (ptpn11) also known as SHP2 (SH2-domain containing phosphatase-2)

This is an example of another phosphatase in which a mutation leads to cancer. It is downstream and activated by most receptor tyrosine kinases (RTKs) involved in activating the MAPK pathway, with its ultimate links to the nucleus and activation of gene transcription.

Figure \(\PageIndex{18}\) shows an interactive iCn3D model of Non-receptor Protein Tyrosine Phosphatase SHP2 in Complex with Allosteric Inhibitor Pyrazolo-pyrimidinone 5 (6MDB)

Figure \(\PageIndex{18}\): Non-receptor Protein Tyrosine Phosphatase SHP2 in Complex with Allosteric Inhibitor Pyrazolo-pyrimidinone 5 (6MDB). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?5WCzu8HeY7JrEVyw5

The phosphatase domain is shown in gray. The N- and C-terminal SH2 domains are shown in green and blue, respectively. The allosteric inhibitor is shown in spacefill and CPK colors. The P-loop in the catalytic domain is shown in red, with Cys 459 (the active site nucleophile) and R465 (a stabilizer of the phosphate in the complex) depicted in sticks, colored according to CPK, and labeled. The bound inhibitor is especially interesting as it binds at an allosteric site. As mentioned above, it is very challenging to design specific inhibitors that target only one PTP, given their common active sites and mechanisms of action. Figure \(\PageIndex{19}\) shows multiple features of SHP2.

Figure \(\PageIndex{19}\):. SH2-domain containing phosphatase-2 SHP2. Köhn ibid.

Panel (A) shows how SHP2, recruited to phosphorylated RTKs, activates the MAPK pathway. The dotted line indicates multiple steps. Upon receptor activation, SHP2 is recruited in different ways to activate the MAPK pathway.

Panel (B) shows that in the inactive state, the N-terminal SH2 domain (green) blocks access to the active site. When the N- and C-terminal domains bind pY residues in a single pY-protein, two pY-proteins, or pY residues on its C-terminal tail, a conformational change ensues, opening the active site (5EHR).

Panel (C) shows how another allosteric inhibitor keeps the protein in a closed state (5EHR).

Panel (D) shows how SHP2 can decrease T-cell responses through the MHC:Tcell receptor (TCR) complex. Tumor cells express a ligand called PD-L1, which binds to the PD1 receptor on the T cell surface. After binding, SHP2 is recruited to PD1, decreasing T cell activation. This is undesirable, as it hinders the immune response to the cancer cell.

Dual Specificity Phosphatases (DUSPs)

Another important phosphatase is phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN (phosphatase and tensin homolog). Dual-specificity protein phosphatase hydrolyzes pTyr- as well as pSer- and pThr-phosphoesters in target proteins. They do not require divalent metal cations and are structurally closer to protein tyrosine phosphatases. They have an active site cysteine in a P-loop also containing arginine. In addition, they are lipid phosphatases that remove phosphate from the inositol ring of phosphatidylinositol derivatives. Both impact many signaling pathways. Its activity as a lipid phosphatase makes it a tumor suppressor protein, as it inhibits the PI3K-AKT/PKB signaling pathway by dephosphorylating phosphoinositides. Hence, it modulates both AKT and mTOR pathways.

The domain structure of PTEN is shown in Figure \(\PageIndex{20}\).

Figure \(\PageIndex{20}\: Structure and regulation of PTEN Chen et al. Endocrinol., 09 July 2018 | https://doi.org/10.3389/fendo.2018.00338. Creative Commons Attribution License (CC BY)

The C2 domain enables phospholipid binding. Multiple post-translational modification sites are indicated. The PEST motif is a sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T) and bounded by positively charged amino acids (Lys, Arg, or His) that act as signals for protein degradation. The PDZ domain, often found at the C-terminal of signaling proteins, acts as a scaffolding site for interaction with other signaling proteins. In the next chapter section, we will consider redox signaling, for which PTEN is a great example. Disulfide formation (in a more oxidizing environment) between the nucleophilic Cys 124 and a nearby Cys 71 (figure above) inhibits PTEN phosphatase activity.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of an AlphaFold computational model of full-length human PTEN (Uniprot P60484).

Figure \(\PageIndex{21}\): AlphaFold computational model of full-length human PTEN (Uniprot P60484). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...1nbf3sDfhvMZ19

The phosphatase (PTPase) domain is shown in blue, and the C2 domain is in orange. The P-loop is shown in red, with the active site cysteine (Cys 124) and R130 in colored sticks and labeled. The backbone of the highly extended intrinsically disordered C-terminus region is shown in gray. It contains the clustered residues Ser 380, Thr 382, Thr 383, and Ser 385 (shown in colored sticks and labeled) that are sites for phosphorylation by activated kinases.

Figure \(\PageIndex{22}\) shows key molecules dephosphorylated by PTEN, including the lipid PIP3, as well as Thr 308 and Ser 473 on AKT.

Figure \(\PageIndex{22}\): A model of how PTEN suppresses the activity of Akt. Bu, L., Wang, H., Pan, Ja. et al. PTEN suppresses tumorigenesis by directly dephosphorylating Akt. Sig Transduct Target Ther 6, 262 (2021). https://doi.org/10.1038/s41392-021-00571-x. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

When PTEN dephosphorylates Akt1, it inhibits AKT activity and effectively antagonizes the main PTEN-PIP3-PDK1-Akt pathway. PTEN is considered a tumor suppressor for this reason. Mutations in PTEN are hence associated with cancer.

Summary

This chapter delves into the critical role of protein phosphatases in reversing protein phosphorylation—a key mechanism by which cells regulate signal transduction and metabolic pathways. While protein kinases add phosphate groups to serine, threonine, or tyrosine residues to modulate protein activity, phosphatases remove these phosphate groups through hydrolytic reactions. The chapter highlights the following major points:

• Reversible Phosphorylation as a Regulatory Mechanism:
  Phosphorylation acts as an on/off switch in cell signaling, and its reversal via dephosphorylation is essential for fine-tuning cellular processes. This dynamic balance controls everything from enzyme activity to gene expression.

• Classification of Phosphatases:
  The text outlines three primary families:

  • Serine/Threonine Phosphatases (PPPs and PPMs):
    Enzymes such as PP1, PP2A, and PP2B (calcineurin) dephosphorylate the majority of pSer and pThr residues. These enzymes often operate as part of a holoenzyme complex where a catalytic subunit associates with diverse regulatory subunits to confer substrate specificity and proper cellular localization.
  • Protein Tyrosine Phosphatases (PTPs):
    These phosphatases, which include receptor-like and intracellular forms (such as PTP1B and low molecular weight PTPs), target phosphorylated tyrosine residues. Their conserved active site features, including a critical cysteine in a CX₅R motif and a supporting WPD loop, facilitate dephosphorylation.
  • Dual Specificity Phosphatases (DUSPs):
    Exemplified by PTEN, these enzymes are capable of dephosphorylating both tyrosine and serine/threonine residues. PTEN’s additional role as a lipid phosphatase, which dephosphorylates phosphatidylinositol derivatives, positions it as a crucial tumor suppressor by antagonizing the PI3K-AKT pathway.

• Catalytic Mechanisms and Structural Insights:
  The catalytic action of these phosphatases often depends on divalent metal ions (e.g., Mn²⁺ or Mg²⁺) that facilitate the generation of a nucleophilic hydroxide ion from water, enabling an SN2 attack on the phosphate group. Structural models (accessible via interactive tools) illustrate how active sites are organized and reveal the subtle differences between phosphatase families that dictate substrate specificity.

• Regulatory Subunits and Targeting:
  For serine/threonine phosphatases like PP1, regulatory subunits—such as glycogen-targeting subunits (GM in muscle and GL in liver) and nuclear targeting proteins (e.g., PNUTS)—play a pivotal role in localizing the enzyme and directing it toward specific substrates. The binding motifs, such as the conserved RVxF sequence, are key for the interaction between the catalytic and regulatory components.

• Physiological and Clinical Implications:
  The chapter underscores how phosphatases regulate essential processes such as glycogen metabolism (by modulating enzymes like glycogen synthase and phosphorylase) and signal transduction pathways critical for cell growth and survival. Dysregulation of phosphatase activity, as seen in mutations affecting PTEN or PTPs, is closely linked to cancer and other diseases, emphasizing their importance as therapeutic targets.

In summary, the chapter provides a comprehensive overview of how protein phosphatases act as essential counterbalances to kinases, ensuring that phosphorylation-dependent signaling events are reversible, precisely regulated, and appropriately localized within the cell. This understanding is crucial not only for appreciating basic cellular regulation but also for developing strategies to modulate these pathways in disease treatment.