Protein kinases and phosphatases jointly regulate the phosphorylation states of proteins, thereby affecting cellular signaling, metabolism, proliferation, and stress responses. Phosphorylation or dephosphorylation does not directly equate to protein activation or inactivation, and its effect depends on the specific modification site and signaling context. Disruption of phosphorylation balance can lead to persistent abnormal signaling and has become an important intervention target in multiple diseases.

Keywords: protein phosphorylation; protein kinase; protein phosphatase; tyrosine kinase; serine/threonine kinase; SHP2; PTEN; signal transduction; targeted therapy; phosphorylation detection

1 Dynamic Balance Between Protein Phosphorylation and Dephosphorylation

1.1 Protein Kinase-Mediated Phosphorylation

Most serine/threonine and tyrosine protein kinases use adenosine triphosphate (ATP) as the phosphate-group donor and transfer the terminal γ-phosphate group of ATP to specific amino acid residues in substrate proteins. In eukaryotic cells, phosphorylation mainly occurs on serine, threonine, and tyrosine residues. In bacterial two-component systems, a sensor histidine kinase generally uses ATP to autophosphorylate a histidine residue and subsequently transfers the phosphoryl group to an aspartate residue in a response regulator. Introduction of a phosphate group can alter local charge distribution, protein conformation, interaction interfaces, and subcellular localization, thereby regulating protein function.

1.2 Protein Phosphatase-Mediated Dephosphorylation

Protein phosphatases acting on phosphorylated serine, threonine, and tyrosine residues remove phosphate groups by hydrolyzing phosphomonoester bonds and restore free hydroxyl groups. Phosphohistidine and phosphoaspartate contain different types of phosphate linkages, and their dephosphorylation does not involve restoration of a free hydroxyl group. Dephosphorylation is not a passive reversal of phosphorylation but an active process jointly controlled by specific phosphatases, regulatory subunits, scaffold proteins, and subcellular localization. Phosphatases can terminate kinase signaling or promote protein activation by removing inhibitory phosphate groups.

1.3 Coordinated Regulation by Kinases and Phosphatases

The phosphorylation level of intracellular proteins depends on the rate at which kinases introduce phosphorylation signals and the rate at which phosphatases remove phosphate groups. Increased kinase activity, reduced phosphatase activity, or altered substrate localization can cause phosphorylation signals to accumulate, whereas kinase inhibition or excessive phosphatase activation may weaken signaling. Cells control the intensity, duration, and spatial range of signals through kinase-phosphatase pairs, negative-feedback loops, and scaffold complexes.

2 Whether Phosphorylation Represents Protein Activation and How to Determine It

2.1 Phosphorylation Does Not Necessarily Indicate Activation

Phosphorylation may promote protein activation, inhibit protein activity, or facilitate protein degradation. For example, dual phosphorylation at specific sites within the ERK activation loop enhances ERK catalytic activity, whereas phosphorylation of certain cyclin-dependent kinases at inhibitory sites reduces their activity. Phosphorylation at different sites of the same protein may also produce opposing effects. Therefore, increased phosphorylation alone cannot be used to conclude that a protein has been activated.

2.2 Dephosphorylation Does Not Necessarily Indicate Inactivation

Dephosphorylation can terminate signaling or relieve inhibition. Cell division cycle 25 phosphatases remove inhibitory phosphate groups from CDK1, thereby promoting CDK1 activation and entry into mitosis, whereas dual-specificity phosphatases remove phosphate groups from the ERK activation loop and terminate MAPK signaling. Therefore, the effect of dephosphorylation must be interpreted according to the specific substrate and modification site.

2.3 Comprehensive Assessment of Protein Activation

To determine whether a protein is truly activated, site-specific phosphorylation, total protein expression, catalytic activity, downstream substrates, subcellular localization, and functional phenotypes should be analyzed together. A protein or signaling pathway can be considered reliably activated only when activating phosphorylation, downstream signaling, and cellular functional changes are mutually consistent.

Table 1 Major Methods for Evaluating Protein Phosphorylation Status and Activity

3 Classification, Structure, and Signal-Writing Mechanisms of Protein Kinases

3.1 Major Types of Protein Kinases

(1) Serine/Threonine Protein Kinases

Serine/threonine protein kinases mainly catalyze phosphorylation of serine or threonine residues in substrates. Representative members include protein kinase A, protein kinase C, calcium/calmodulin-dependent protein kinases, RAF, AKT, mTOR, and ROCK.

(2) Protein Tyrosine Kinases

Protein tyrosine kinases mainly catalyze phosphorylation of tyrosine residues and can be divided into receptor and nonreceptor types. EGFR, VEGFR, PDGFR, and MET are receptor tyrosine kinases, whereas SRC, JAK, and ABL are nonreceptor tyrosine kinases.

(3) Dual-Specificity Protein Kinases

Dual-specificity kinases can catalyze phosphorylation of more than one class of amino acid residue. MEK1 and MEK2 simultaneously phosphorylate threonine and tyrosine residues within the ERK activation loop and are typical dual-specificity kinases in the MAPK cascade.

3.2 Catalytic Domain of Protein Kinases

A typical protein kinase catalytic domain consists of approximately 250-300 amino acids and can be divided into a smaller N-terminal lobe and a larger C-terminal lobe, with an ATP-binding cleft formed between them. The N-terminal lobe mainly participates in ATP positioning, whereas the C-terminal lobe is involved in substrate recognition and phosphate-group transfer. The activation loop, DFG motif, and catalytic loop jointly determine whether the kinase can adopt a catalytically active conformation.

3.3 Mechanisms of Protein Kinase Activation

Protein kinases can be activated through ligand binding, receptor dimerization, activation-loop phosphorylation, second-messenger binding, or dissociation of regulatory subunits. Receptor tyrosine kinases generally undergo dimerization and transphosphorylation after ligand binding. PKA is regulated by cyclic adenosine monophosphate. The activation requirements differ among PKC subfamilies: conventional PKCs are generally regulated by diacylglycerol, Ca²⁺, and membrane phospholipids; novel PKCs depend on diacylglycerol but not Ca²⁺; and atypical PKCs depend on neither diacylglycerol nor Ca²⁺. CaMKs are activated by Ca²⁺–calmodulin complexes.

4 Classification, Substrate Selection, and Signal Resetting by Protein Phosphatases

4.1 Serine/Threonine Protein Phosphatases

(1) PPP Family

The phosphoprotein phosphatase family includes PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7. Their catalytic subunits generally need to bind regulatory subunits or scaffold proteins to recognize specific substrates and localize to the plasma membrane, nucleus, cytoskeleton, or other cellular structures.

(2) PPM Family

The metal-dependent protein phosphatase family is represented by PP2C-type proteins, and their catalytic activity generally depends on Mg²⁺ or Mn²⁺. PPM-family members are generally independent of the regulatory subunits commonly used by the PPP family and instead control substrate selection through their intrinsic structure, expression location, and stress status.

4.2 Protein Tyrosine Phosphatases

Protein tyrosine phosphatases include receptor-type, nonreceptor-type, and dual-specificity phosphatases. Some members directly remove tyrosine phosphate groups from receptors or intracellular signaling proteins, whereas dual-specificity phosphatases can act on phosphorylated serine, threonine, and tyrosine residues.

4.3 Substrate Specificity of Phosphatases

The catalytic domains of many phosphatases have limited direct substrate selectivity, and their specificity depends more heavily on regulatory subunits, scaffold proteins, substrate-binding sequences, and subcellular localization. Changes in phosphatase-complex composition can cause abnormalities in specific signaling pathways even when the expression level of the catalytic subunit remains unchanged.

4.4 Functional Differences Between PTEN and SHP2

PTEN is a tumor-suppressor protein with lipid phosphatase activity that converts phosphatidylinositol-3,4,5-trisphosphate into phosphatidylinositol-4,5-bisphosphate, thereby restricting AKT recruitment and activation. SHP2 is encoded by the PTPN11 gene and, although classified as a protein tyrosine phosphatase, promotes signal transmission from receptor tyrosine kinases to the RAS-MAPK pathway. Phosphatases may suppress abnormal signaling or function as positive regulators required for proliferative signaling.

5 Typical Signaling Networks Jointly Regulated by Kinases and Phosphatases

5.1 EGFR-RAS-MAPK Signaling Axis

Binding of EGF to EGFR promotes receptor dimerization and tyrosine phosphorylation, which recruits adaptor proteins and activates RAS, RAF, MEK, and ERK. SHP2 promotes RAS activation and signal propagation downstream, certain protein tyrosine phosphatases reduce EGFR phosphorylation, and the DUSP family removes phosphate groups from the ERK activation loop. Output from this pathway is jointly determined by kinases such as EGFR, RAF, and MEK and phosphatases such as SHP2, DUSP, and PP2A.

Figure 1 EGFR-mediated downstream signaling network

5.2 PI3K-AKT-mTOR Signaling Axis

After receptor tyrosine kinase activation, PI3K can be recruited and promote PIP3 production. PIP3 subsequently recruits PDK1 and AKT to the plasma membrane, allowing AKT phosphorylation and activation of mTOR-associated signaling. PTEN restricts AKT membrane recruitment by removing a phosphate group from PIP3, whereas protein phosphatases such as PP2A regulate the phosphorylation states of AKT and other downstream proteins. Increased PI3K or AKT activity and loss of PTEN function can both cause persistent activation of this pathway.

5.3 RhoA-ROCK-Myosin Signaling Axis

Activated RhoA promotes ROCK activation. ROCK phosphorylates myosin light chain and the regulatory subunit of myosin phosphatase, increasing myosin light-chain phosphorylation and enhancing actin-myosin contraction. Myosin light-chain phosphatase removes the corresponding phosphate groups and weakens cellular contraction. This balance participates in regulation of vascular tone, cell migration, adhesion, and tissue fibrosis.

5.4 Glycogen Phosphorylase Regulatory Axis

Glycogen phosphorylase participates in glycogen breakdown. After Ser14 is phosphorylated by phosphorylase kinase, the enzyme can be converted from the low-activity phosphorylase b form into the high-activity phosphorylase a form. Protein phosphatase 1 removes the phosphate group from Ser14 and restores the low-activity state. This kinase-phosphatase balance determines the rate of glycogen breakdown in the liver and muscle and affects blood glucose and energy supply.

6 Disruption of Phosphorylation Balance and Disease Development

6.1 Persistent Kinase Activation

Mutations, gene amplification, or excessive ligand production involving receptor tyrosine kinases can cause persistent activation of EGFR, HER2, MET, VEGFR, and related signaling pathways. Abnormalities in intracellular kinases such as RAF, AKT, JAK, and ROCK can also bypass normal regulation and enhance cellular proliferation, survival, contraction, or inflammatory signaling.

6.2 Loss of Inhibitory Phosphatase Function

Loss of PTEN can cause PIP3 accumulation and enhanced AKT-mTOR signaling, whereas reduced PP2A function can maintain multiple kinase substrates in a phosphorylated state. Loss of certain protein tyrosine phosphatases can also prolong receptor tyrosine kinase signaling. These abnormalities are not caused by changes in the kinases themselves but by reduced dephosphorylation capacity.

6.3 Abnormal Activation of Signal-Promoting Phosphatases

Some phosphatases promote signal transmission. SHP2 facilitates RAS-MAPK signaling, and activating PTPN11 mutations or abnormal SHP2 activation can enhance cell proliferation and survival. Therefore, phosphatases should not be uniformly regarded as signaling inhibitors, because their functions depend on the substrate, pathway position, and cellular context.

6.4 Signal Reprogramming and Drug Tolerance

After prolonged inhibition of a single kinase, cells can restore downstream signaling through feedback activation of upstream receptors, enhancement of bypass kinases, altered phosphatase expression, or reorganization of scaffold complexes. For example, MET or HER2 signaling may increase after EGFR inhibition, whereas MEK inhibition may also induce feedback activation of receptor tyrosine kinases. Resistance studies therefore need to evaluate both kinase and phosphatase networks.

7 Targeted Intervention Strategies for Protein Kinases

7.1 ATP-Competitive Inhibition

ATP-competitive inhibitors occupy the ATP-binding pocket of a kinase and prevent ATP from participating in phosphate-group transfer. Because ATP-binding regions show a certain degree of similarity among different kinases, these drugs must balance inhibitory potency with target selectivity.

7.2 Covalent and Allosteric Inhibition

Covalent inhibitors form stable covalent bonds with specific amino acid residues near the kinase-binding pocket and prolong target occupancy. Allosteric inhibitors bind regulatory regions outside the ATP pocket and stabilize an inactive conformation or prevent conformational transitions. Both strategies can improve selectivity for specific kinase conformations or mutant forms.

7.3 Dual-Target and Multitarget Inhibition

Dual-target or multitarget compounds can simultaneously block multiple interconnected kinase nodes and reduce certain forms of bypass compensation, but they may also increase off-target effects. Studies should simultaneously evaluate the principal targets, downstream signaling, and cellular functions rather than determining drug activity solely from inhibition of a single target.

7.4 Kinase Activation and Functional Restoration

Not all disease studies aim to inhibit kinases. In energy-metabolism and stress-regulation research, protective kinases such as AMPK can also be activated to restore energy homeostasis. The choice between kinase activators and kinase inhibitors depends on the functional direction of the target protein in the specific disease context.

8 Drug-Development Challenges and Emerging Intervention Strategies for Protein Phosphatases

The catalytic centers of phosphatases must recognize negatively charged phosphorylated substrates. Compounds that directly bind the active site are therefore generally highly polar and may exhibit poor cell-membrane permeability and low bioavailability. Catalytic structures may also be highly similar among phosphatases of the same family, increasing the difficulty of achieving selective inhibition.

8.2 Allosteric Inhibition of Phosphatases

Allosteric inhibitors bind regulatory regions outside the catalytic site and stabilize an inactive phosphatase conformation or prevent conformational transitions. This strategy avoids direct competition with the highly polar catalytic pocket and can exploit structural differences among phosphatases to improve selectivity.

8.3 Restoration of Inhibitory Phosphatase Function

For phosphatases with signal-suppressing functions, such as PTEN and PP2A, the intervention objective is generally not further inhibition but restoration of their expression, complex assembly, or catalytic function. Relevant strategies include blocking inhibitory proteins, stabilizing regulatory subunits, and promoting formation of functional phosphatase complexes.

8.4 Targeting Phosphatase Complexes and Localization

The substrate specificity of phosphatases is often determined by regulatory subunits and subcellular localization. Interfering with interactions between phosphatases and regulatory subunits, scaffold proteins, or substrates can selectively alter a specific signal without completely inhibiting the catalytic subunit, thereby reducing nonspecific effects caused by widespread alteration of cellular phosphorylation networks.

9 Products Related to Kinase, Phosphatase, and Phosphorylation Research

9.1 Protein Kinase Signaling Modulators

9.2 Protein Phosphatase Signaling Modulators

9.3 Kinase and Phosphatase Activity Detection Products

9.4 Products for Protein Extraction and Preservation of Phosphorylation Status

9.5 Kinase Compound-Screening Products

Protein kinases and phosphatases jointly determine the intensity and duration of phosphorylation signals. Related studies should integrate specific phosphorylation sites, upstream and downstream pathways, and functional phenotypes to comprehensively evaluate target status and intervention effects.

References

[1] Nishi H, Shaytan A, Panchenko AR. Physicochemical mechanisms of protein regulation by phosphorylation. Front Genet. 2014;5:270.

[2] Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell. 2009;139(3):468-484.

[3] Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol. 2006;7(11):833-846.

[4] Cohen P. Protein kinases—the major drug targets of the twenty-first century. Nat Rev Drug Discov. 2002;1(4):309-315.

For more related articles, please see below:

[1] Protein kinase C isoform analysis experiment

[2] Phosphorylated and Sulfonated Peptides

[3] Protease & Phosphatase Inhibitor Cocktails: Research Applications and Principles