Protein tyrosine kinases (PTKs) transfer the γ-phosphate group of ATP to tyrosine residues on protein substrates and participate in the regulation of cell proliferation, differentiation, migration, metabolism, and survival. PTKs mainly include receptor tyrosine kinases (RTKs) and nonreceptor tyrosine kinases (NRTKs). Together, they form an important signaling network for growth factor, immune receptor, integrin, and tumor microenvironment signals.

Keywords: protein tyrosine kinase; RTK; NRTK; EGFR; FGFR; VEGFR; JAK; Src; Abl; tumor targeted therapy

1 Basic Logic of PTK/RTK Signaling Research

1.1 Signaling Significance of Protein Tyrosine Phosphorylation

Protein tyrosine phosphorylation is not merely a protein modification, but a highly reversible form of signal encoding. After tyrosine residues are phosphorylated, protein conformation, enzymatic activity, subcellular localization, and protein interactions may change, and binding sites are generated for signaling proteins containing SH2 domains or PTB domains.

(1) Kinase level

PTKs catalyze tyrosine phosphorylation and often convert external stimuli into intracellular signaling outputs. RTKs are located on the cell membrane and can directly sense ligands. NRTKs are mostly located in the cytoplasm, at the inner side of the membrane, or in cytoskeleton-associated regions, where they connect receptors with downstream pathways.

(2) Substrate level

Phosphorylated substrates include receptors themselves, adaptor proteins, transcription factors, cytoskeletal proteins, metabolic regulatory proteins, and immune signaling molecules. Different substrate combinations determine different outputs such as cell proliferation, migration, differentiation, or immune activation.

(3) Negative regulation level

Protein tyrosine phosphatases, receptor internalization, ubiquitin-mediated degradation, and feedback inhibitors can limit PTK signal intensity. If negative regulation fails, RTK or NRTK signaling may remain persistently activated, forming disease-associated abnormal signaling states.

1.2 Functional Differences Between RTKs and NRTKs

PTKs can be divided into transmembrane receptor-type kinases and nonreceptor-type kinases. They differ in spatial localization and activation mode, but often connect with each other in downstream signaling networks.

(1) RTKs

RTKs are transmembrane glycoproteins. Their typical structure includes an extracellular ligand-binding region, a transmembrane region, and an intracellular tyrosine kinase domain. After ligand binding, receptor dimerization or conformational change occurs, and the intracellular kinase domains undergo trans-autophosphorylation, providing binding sites for downstream signaling proteins.

(2) NRTKs

NRTKs do not have transmembrane structures and include Src, JAKs, Abl, FAK, Syk, BTK, and others. They participate in RTK, cytokine receptor, integrin, GPCR, and immune receptor signaling and serve as important relay nodes in cell migration, immune activation, inflammation, and tumor signaling.

(3) Signal interpretation

Increased RTK expression does not equal RTK activation, and increased total NRTK protein does not equal enhanced pathway activity. Experiments should focus on site-specific phosphorylation, kinase activity, downstream pathways, and functional phenotypes, rather than explaining mechanisms only by total protein expression.

Table 1 Major Types of PTKs and Research Focuses

2 RTK Activation Mechanisms and Downstream Pathways

2.1 Ligand Binding and Receptor Dimerization

(1) Mechanistic core

Most RTKs undergo ligand-induced dimerization or receptor conformational rearrangement, bringing two intracellular kinase domains close together and allowing trans-autophosphorylation. Autophosphorylation sites enhance receptor kinase activity and also form binding platforms for downstream signaling proteins.

(2) Experimental indicators

Common indicators include ligand-induced receptor phosphorylation, receptor dimerization, membrane localization, receptor internalization, time-course phosphorylation, and dose-response curves.

(3) Result interpretation

Rapid elevation of p-RTK after ligand stimulation usually indicates receptor activation. If total receptor expression is high but phosphorylation does not increase, possible explanations include lack of ligand, abnormal receptor localization, inactivation of the kinase domain, or negative feedback inhibition.

2.2 Phosphotyrosine Binding and Signal Branching

(1) Mechanistic core

After RTK autophosphorylation, proteins containing SH2 or PTB domains can recognize phosphotyrosine sites. After different adaptor proteins are recruited, signals branch into pathways such as Ras/MAPK, PI3K/AKT, PLCγ/PKC, JAK/STAT, and Src/FAK.

(2) Experimental indicators

Recruitment or phosphorylation of molecules such as Grb2, Shc, Gab, IRS, PI3K p85, PLCγ, STAT, Src, and FAK can be detected and validated together with downstream p-ERK, p-AKT, p-STAT, and p-PLCγ.

(3) Result interpretation

The same RTK can activate multiple pathways simultaneously. If only p-ERK or p-AKT is detected, signaling branches may be missed. A more reliable design is to detect receptor phosphorylation, adaptor protein recruitment, and downstream pathway output simultaneously.

Figure 1 Schematic diagram of MAPK, PI3K-Akt, PLC-PKC and JAK/STAT pathways downstream of RTK activation

2.3 RTK Signal Termination and Negative Feedback

(1) Mechanistic core

RTK signaling is usually restricted by receptor internalization, lysosomal degradation, phosphatase-mediated dephosphorylation, ubiquitination, and downstream negative feedback. Persistent RTK activation can lead to abnormal proliferation or adaptive drug resistance.

(2) Experimental indicators

Receptor internalization, Cbl-mediated ubiquitination, membrane receptor level, total receptor degradation, protein tyrosine phosphatase activity, and feedback regulatory molecules such as DUSP, Sprouty, and SOCS are commonly detected.

(3) Result interpretation

Reduced RTK phosphorylation does not necessarily mean that a drug directly inhibits the kinase; it may also result from receptor internalization or degradation. To determine whether a drug is a kinase inhibitor, in vitro kinase activity, site-specific phosphorylation, and changes in total receptor protein should be evaluated together.

Table 2 Major Downstream Pathways After RTK Activation

3 Major RTK Families and Research Directions

3.1 EGFR Family

(1) Mechanistic core

The EGFR family participates in epithelial cell proliferation, differentiation, migration, and tissue repair. EGFR, HER2, HER3, and HER4 can form homo- or heterodimers, and different combinations generate different signaling intensities and pathway biases.

(2) Research indicators

EGFR/HER2 expression, p-EGFR, p-HER2, p-ERK, p-AKT, receptor internalization, proliferation, migration, and sensitivity to EGFR inhibitors are commonly detected.

(3) Result interpretation

Increased EGFR expression does not necessarily indicate EGFR dependence. EGFR-driven function is better supported only when EGFR inhibition reduces p-ERK/p-AKT and is accompanied by reduced proliferation or migration. When HER2 amplification or HER3 involvement is present, receptor heterodimerization and bypass signaling should be considered.

3.2 FGFR Family

(1) Mechanistic core

The FGFR family participates in development, angiogenesis, tissue repair, and tumor progression. FGF ligands usually require heparin or heparan sulfate to form stable complexes and activate downstream MAPK, PI3K, and PLCγ signaling through FGFR.

(2) Research indicators

FGFR expression, FGFR fusion, amplification or mutation, p-FGFR, p-FRS2, p-ERK, p-AKT, cell proliferation, and differentiation markers can be detected.

(3) Result interpretation

FGFR abnormalities may arise from amplification, fusion, mutation, or ligand-dependent activation. Detection of total FGFR protein alone cannot distinguish the driving mechanism. Fusion or mutant FGFR is more likely to show ligand-independent activation.

3.3 PDGFR and VEGFR Families

(1) Mechanistic core

PDGFR mainly participates in mesenchymal cell proliferation, migration, and tissue remodeling, while VEGFR is a core RTK for angiogenesis. The VEGF-VEGFR axis can promote endothelial cell proliferation, migration, tube formation, and increased vascular permeability.

(2) Research indicators

For PDGFR, p-PDGFR, fibroblast activation, migration, and fibrosis markers are commonly detected. For VEGFR, p-VEGFR2, endothelial cell migration, tube formation, angiogenesis, and hypoxia-related signals are commonly detected.

(3) Result interpretation

Enhanced VEGFR signaling usually suggests active angiogenesis, but endothelial cell function and tissue vascular density should be considered. In tumors, VEGFR inhibition may affect angiogenesis and may also produce indirect effects by altering hypoxia and the immune microenvironment.

3.4 Insulin Receptor and IGF1R

(1) Mechanistic core

The insulin receptor and IGF1R connect metabolic signaling with cell growth. The insulin receptor is more biased toward regulating glucose uptake, glucose and lipid metabolism, and metabolic homeostasis, while IGF1R is more commonly associated with growth, survival, and anti-apoptotic responses.

(2) Research indicators

IR/IGF1R phosphorylation, IRS1/2, p-AKT, GLUT4 translocation, glucose uptake, mTOR signaling, and cell survival are commonly detected.

(3) Result interpretation

Increased AKT may suggest enhanced insulin/IGF signaling, but metabolic indicators should also be considered. If p-AKT increases but glucose uptake does not change significantly, the signal may be biased toward survival or growth branches rather than typical metabolic effects.

3.5 Trk, MET, ALK, RET, and Eph Receptors

(1) Trk receptors

TrkA, TrkB, and TrkC recognize different neurotrophic factors and regulate neuronal survival, axonal growth, synaptic maturation, and pain signaling. p-Trk, p-ERK, p-AKT, neurite outgrowth, and neuronal survival are commonly detected.

(2) MET

The MET receptor is activated by HGF and participates in cell migration, invasion, epithelial-mesenchymal transition, and tissue repair. In tumors, MET amplification, overexpression, or exon skipping mutations can drive invasion and drug resistance.

(3) ALK and RET

ALK and RET can form persistently activated signals through gene rearrangements, fusions, or mutations and are important targets in some lung cancers, thyroid cancers, and neural-related tumors.

(4) Eph receptors

Eph receptors and ephrin ligands mediate cell contact-dependent signaling and participate in axon guidance, vascular patterning, cell positioning, and tumor migration. Eph signaling is bidirectional and spatially dependent, so interpretation should consider cell contact and microenvironmental structure.

4 NRTKs and Expansion of Receptor Signaling

4.1 Src Family Kinases

(1) Mechanistic core

Src family kinases crosstalk with RTK, integrin, GPCR, and immune receptor signaling to regulate cytoskeleton, adhesion, migration, and invasion. Src activation often enhances tumor cell motility and metastatic potential.

(2) Experimental indicators

p-Src, FAK/Paxillin phosphorylation, cell migration, invasion, adhesion, stress fibers, and cell morphological changes are commonly detected.

(3) Result interpretation

Increased p-Src should be interpreted together with migration or invasion phenotypes. If Src inhibition reduces p-FAK and inhibits cell migration, the Src-FAK axis may be involved in the motility phenotype.

4.2 JAK Family Kinases

(1) Mechanistic core

JAKs mainly connect cytokine receptors with STAT transcription factors and have core roles in immunity, inflammation, hematopoiesis, and the tumor microenvironment. After activation, JAK can phosphorylate STAT, promoting STAT dimerization, nuclear translocation, and target gene expression.

(2) Experimental indicators

p-JAK, p-STAT1/3/5, STAT nuclear translocation, inflammatory factors, cytokine stimulation responses, and sensitivity to JAK inhibitors are commonly detected.

(3) Result interpretation

Increased p-STAT usually indicates active JAK/STAT signaling, but it may also be indirectly driven by RTKs or other kinases. If JAK inhibitors reduce p-STAT and alter inflammatory or proliferative phenotypes, JAK dependence is better supported.

4.3 Abl Kinases

(1) Mechanistic core

Abl kinases participate in cytoskeletal regulation, DNA damage response, and proliferation control. The BCR-ABL fusion protein forms constitutive kinase activity and is an important driver of chronic myeloid leukemia.

(2) Experimental indicators

BCR-ABL, p-Abl, CrkL phosphorylation, cell proliferation, apoptosis, colony formation, and sensitivity to ABL inhibitors are commonly detected.

(3) Result interpretation

Sensitivity of BCR-ABL-positive cells to ABL inhibitors usually indicates dependence on the fusion kinase. If drug tolerance emerges, mutations in the ABL kinase domain, bypass pathways, and clonal evolution should be examined.

4.4 FAK, Syk, and BTK

(1) FAK

FAK connects integrin, cell adhesion, and RTK signaling and often participates in tumor migration, invasion, and adaptation to the mechanical microenvironment.

(2) Syk

Syk is important in immune receptor signaling and participates in B cell receptor, Fc receptor, and inflammation-related signaling.

(3) BTK

BTK is an important NRTK in B cell receptor signaling and participates in B cell activation, differentiation, and hematologic malignancies.

(4) Result interpretation

These NRTKs often function as downstream signal amplifiers of receptors. Research should combine receptor stimulation, kinase phosphorylation, downstream transcriptional responses, and cellular function rather than relying only on total protein expression.

Table 3 NRTKs and Major Signaling Functions

5 PTK/RTK Abnormalities and Disease Mechanisms

5.1 RTK Dysregulation in Tumors

RTK dysregulation is an important mechanism of tumorigenesis and tumor progression and mainly includes genomic rearrangements, autocrine activation, overexpression, and gain-of-function or loss-of-function mutations.

(1) Genomic rearrangement

ALK, RET, NTRK, FGFR, and other genes can form constitutive kinase activity through fusion or rearrangement. These abnormalities usually do not depend on exogenous ligands and can directly drive downstream MAPK, PI3K, or STAT pathways.

(2) Autocrine activation

Tumor cells may express both RTKs and their ligands, forming autocrine loops that keep receptors chronically activated. This mechanism is common in growth factor-rich tumor microenvironments.

(3) Overexpression

Overexpression of receptors such as EGFR, HER2, and MET can increase cellular sensitivity to ligands and enhance downstream proliferative and survival signaling. Whether overexpression is functionally driving should be validated through inhibition or knockdown experiments.

(4) Mutation

Gain-of-function mutations can cause sustained receptor activation, while loss-of-function mutations may disrupt normal differentiation, homeostasis, or negative regulation. Mechanistic interpretation of mutations should combine mutation site, kinase activity, downstream signaling, and cellular phenotype.

5.2 Angiogenesis and Ocular Diseases

Abnormally enhanced VEGF/VEGFR signaling can promote pathological angiogenesis and increased vascular permeability and participates in diabetic retinopathy, age-related macular degeneration, and tumor angiogenesis.

(1) Experimental indicators

VEGF, p-VEGFR2, endothelial cell migration, tube formation, vascular permeability, hypoxia markers, and tissue neovascularization are commonly detected.

(2) Result interpretation

Increased VEGF does not necessarily mean that the VEGFR pathway is effectively activated. p-VEGFR2 and vascular functional indicators should be combined for interpretation. Anti-VEGF or VEGFR inhibition research should also consider hypoxic compensation and bypass angiogenic signaling.

5.3 Atherosclerosis and Inflammatory Diseases

RTKs and NRTKs can participate in vascular smooth muscle cell migration, endothelial function changes, macrophage activation, and inflammatory factor release. PDGFR, VEGFR, Src, JAK, and other signals can be associated with vascular remodeling and inflammation amplification.

(1) Experimental indicators

Smooth muscle cell migration, endothelial permeability, inflammatory factors, p-Src, p-JAK/STAT, p-PDGFR, and plaque-related markers are commonly detected.

(2) Result interpretation

Inflammatory pathways and growth factor pathways often crosstalk. If inhibition of a kinase reduces inflammatory factors, it is still necessary to determine whether the effect is directly on immune signaling or indirectly caused by reduced cell migration or tissue injury.

5.4 Psoriasis and Immune Regulation

JAK/STAT, Src family, and some RTK-related signals can participate in keratinocyte proliferation, inflammatory cell recruitment, and cytokine responses. In inflammatory diseases such as psoriasis, PTK signaling often crosstalks with the IL-23/IL-17 axis, TNF signaling, and skin barrier abnormalities.

(1) Experimental indicators

p-JAK, p-STAT, keratinocyte proliferation, inflammatory factors, immune cell infiltration, and skin barrier-related proteins are commonly detected.

(2) Result interpretation

When PTK inhibition reduces inflammation, cytotoxicity and tissue barrier repair should also be observed. If inflammatory factors are reduced but barrier function is impaired, the result should not be simply interpreted as an effective therapeutic mechanism.

6 PTK/RTK-Targeted Intervention Strategies

6.1 Small-Molecule Kinase Inhibitors

(1) Mechanistic core

Small-molecule kinase inhibitors mostly act on the ATP-binding pocket or nearby regulatory sites and inhibit catalytic kinase activity. Their advantage is that they can enter cells and block intracellular kinase activity, but off-target effects and resistance mutations may also occur.

(2) Experimental indicators

Target kinase phosphorylation, downstream p-ERK/p-AKT/p-STAT changes, cell proliferation, apoptosis, colony formation, and kinase selectivity are commonly detected.

(3) Result interpretation

If an inhibitor reduces p-RTK but downstream pathways do not significantly decrease, bypass activation or downstream mutations may exist. If downstream pathways decrease while total receptor levels remain unchanged, the drug may effectively inhibit kinase activity rather than reduce receptor expression.

6.2 Monoclonal Antibodies and Ligand Blockade

(1) Mechanistic core

Monoclonal antibodies can act by blocking ligand binding, preventing receptor dimerization, inducing receptor internalization and degradation, or mediating immune effector functions. Anti-VEGF antibodies reduce VEGFR activation by neutralizing the ligand.

(2) Experimental indicators

Ligand-receptor binding, receptor internalization, decreased p-RTK, cell proliferation, angiogenesis, ADCC-related indicators, and tissue-level efficacy are commonly detected.

(3) Result interpretation

Antibody drugs do not necessarily directly inhibit the kinase domain. If receptor phosphorylation decreases, it is necessary to determine whether the effect results from ligand blockade, receptor internalization, or immune-mediated effects.

6.3 Combination Therapy

(1) Mechanistic core

RTK signaling is highly networked. After inhibition of a single RTK, compensatory bypass pathways often emerge, such as MET, HER3, IGF1R, AXL, or downstream MAPK/PI3K reactivation. The purpose of combination therapy is to block both the driver pathway and resistance bypasses.

(2) Experimental indicators

p-RTK, p-ERK, p-AKT, p-STAT, cell viability, colony formation, resistant-strain sensitivity, and synergistic effects after combination treatment can be detected.

(3) Result interpretation

Combination therapy should not be judged only by short-term decreases in cell viability. It should be determined whether bypass activation is truly reduced, whether resistance emergence is delayed, and whether the therapeutic window is expanded. If combined toxicity is close to the effective concentration, translational value should be evaluated cautiously.

7 PTK/RTK Resistance Mechanisms

7.1 Target Mutations

(1) Mechanistic core

Mutations in kinase domains can reduce inhibitor binding or alter kinase conformation, making drugs unable to effectively inhibit the target. Typical resistance often occurs in the ATP-binding pocket, gatekeeper site, or activation loop region.

(2) Experimental indicators

Target sequencing, p-RTK detection, drug dose-response curves, construction of resistant strains, and structural modeling are commonly performed.

(3) Result interpretation

If resistant cells retain p-RTK and show kinase domain mutations, on-target resistance is suggested. Next-generation inhibitors or allosteric inhibitors can then be evaluated.

7.2 Bypass Pathway Activation

(1) Mechanistic core

Tumor cells can bypass the original target by activating other RTKs or NRTKs, such as MET, HER3, AXL, IGF1R, JAK/STAT, or Src signaling, thereby maintaining downstream ERK/AKT/STAT activity.

(2) Experimental indicators

p-RTK array, p-ERK, p-AKT, p-STAT, bypass receptor expression, ligand secretion, and effects of combined inhibition are commonly detected.

(3) Result interpretation

If the original target is inhibited but p-ERK or p-AKT recovers, bypass RTKs or downstream mutations should be investigated first. Bypass activation-mediated resistance usually requires combined inhibition rather than simply increasing the dose of the original drug.

7.3 Downstream Pathway Mutations

(1) Mechanistic core

Abnormalities in downstream molecules such as RAS, BRAF, PIK3CA, and PTEN can make cells insensitive to upstream RTK inhibition. In this situation, the RTK is inhibited, but downstream proliferative or survival signaling can still be maintained.

(2) Experimental indicators

RAS/RAF/PI3K/PTEN status, p-ERK, p-AKT, drug sensitivity, and responses to downstream pathway inhibitors are commonly detected.

(3) Result interpretation

If RTK phosphorylation decreases but downstream p-ERK or p-AKT does not decrease, resistance may occur downstream of the receptor. Treatment strategies should shift toward downstream nodes or combined blockade.

7.4 Phenotypic Switching and Microenvironmental Adaptation

(1) Mechanistic core

Tumor cells can generate non-genetic resistance through epithelial-mesenchymal transition, enhanced stem cell-like states, immune evasion, hypoxic adaptation, or changes in paracrine factors.

(2) Experimental indicators

EMT markers, tumor sphere formation, invasion, hypoxia markers, cytokines, stromal cell co-culture, and long-term resistance models can be detected.

(3) Result interpretation

If there is no clear target mutation but cell migration, invasion, stemness, or microenvironmental dependence is enhanced, phenotypic adaptive resistance should be considered. This type of resistance often requires combined microenvironmental intervention or multi-pathway therapy.

Table 4 PTK/RTK Resistance Mechanisms and Validation Strategies

Table 5 Representative Products Related to PTK/RTK Signaling Research

Table 6 Product Lines Related to PTK/RTK and Application Directions

Protein tyrosine kinase signaling research should focus on kinase activation status, downstream pathway dependence, and sources of resistance. Only by correlating receptor phosphorylation, driver mechanisms, cellular phenotypes, and drug responses can the roles of PTK/RTK in tumors, vascular abnormalities, metabolic diseases, and inflammatory diseases be accurately determined.

References

[1] Robinson DR, et al. Oncogene. 2000 Nov 20;19(49):5548-5557.

[2] McDonell LM, et al. Hum Mol Genet. 2015 Oct 15;24(R1):R60-R66.

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