p59-Fyn Human

What is p59-Fyn and what are its key structural characteristics?

p59-Fyn is a member of the protein-tyrosine kinase oncogene family, specifically belonging to the Src-family of kinases. It functions as a membrane-associated non-receptor protein tyrosine kinase implicated in cell growth control . The mature protein contains several important domains: a unique N-terminal region (residues 1-84), SH3 domain (residues 85-139), SH2 domain (residues 140-248), and a catalytic tyrosine kinase domain. The recombinant form of p59-Fyn (aa 23-216) including the GST tag shows a molecular weight of approximately 50 kDa on SDS-PAGE . The human p59-Fyn gene (FYN) is identified by Gene ID 2534, with several synonyms including SLK, SYN, and MGC45350 .

What is the physiological role of p59-Fyn in cellular systems?

p59-Fyn plays critical roles in multiple cellular processes. It associates with the p85 subunit of phosphatidylinositol 3-kinase and interacts with the fyn-binding protein, contributing to various signaling pathways . In the nervous system, p59-Fyn is highly expressed in the brain, suggesting potential roles in the sensory nervous network and myelination during early stages of CNS formation . In immune cells, p59-Fyn interacts with the CD3 and eta chains of the T cell receptor (TCR) through its unique N-terminal domain, playing an important role in T cell signaling . The protein can bind other proteins (p82 and p116) through its SH2 and SH3 domains, which may function as substrates or regulators of Fyn activity . Distinct isoforms of p59-Fyn exist due to alternative splicing, suggesting tissue-specific functions .

How should p59-Fyn protein be stored and handled in a laboratory setting?

For optimal stability and activity, p59-Fyn should be stored at -20°C to -80°C, where it remains stable for up to 12 months . The protein is typically supplied in a formulation containing 50 mM Tris-Acetate (pH 7.5), 1 mM EDTA, and 20% Glycerol . It is critical to prevent freeze-thaw cycles as they can lead to denaturation and loss of activity . When working with p59-Fyn, maintain sterile conditions and handle the protein on ice when possible. For experimental applications including ELISA, inhibition assays, and Western Blotting, the protein should be thawed gently and kept at appropriate temperatures specific to each assay protocol . Always follow good laboratory practices, including using dedicated pipettes and sterile consumables to prevent contamination.

What is the significance of dual fatty acylation in p59-Fyn function and association with the T cell receptor?

Dual fatty acylation of p59-Fyn is essential for its proper localization and function. Research has demonstrated that both myristoylation and palmitoylation are required for stable association with the T cell receptor . Specifically, the first 10 residues within the SH4 domain are critical for this interaction. Glycine at position 2 is required for myristoylation, while cysteine at position 3 serves as the main site of palmitoylation . Additionally, lysines at positions 7 and 9 direct efficient myristoylation . Mutation of these key residues (G2A, C3S, K7A, K9A) significantly impairs fatty acylation and consequently disrupts the ability of p59-Fyn to interact with the TCR ζ chain .

Experimental evidence shows that when fatty acylation of p59-Fyn is blocked using inhibitors like 2-hydroxymyristate, the association with the ζ chain is disrupted . This indicates that proper membrane targeting through dual acylation is a prerequisite for stable interaction with TCR components. The importance of these modifications is further underscored by experiments showing that overexpression of N-myristoyltransferase (NMT) can restore fatty acylation levels of K7,9A-Fyn mutants and rescue their ability to interact with the TCR ζ chain .

How do the SH2 and SH3 domains contribute to p59-Fyn functionality in signaling pathways?

The SH2 and SH3 domains of p59-Fyn play crucial roles in mediating protein-protein interactions essential for signaling. The SH2 domain (residues 140-248) recognizes and binds to phosphorylated tyrosine residues on target proteins, while the SH3 domain (residues 85-139) binds to proline-rich sequences . In T cell signaling, functional studies have demonstrated that both Fyn kinase and SH2 domains are required for stable association with the TCR ζ chain . The process involves initial phosphorylation of tyrosine residues in the immune receptor tyrosine-based activation motifs (ITAMs) of the ζ chain by Fyn kinase, followed by binding of the Fyn SH2 domain to these phosphotyrosines .

Mutation studies provide valuable insights into domain functionality. Deletion of the SH2 domain (ΔSH2-Fyn) or mutation of a critical arginine residue in the phosphotyrosine binding pocket (R176K-Fyn) compromises the ability of p59-Fyn to form stable complexes with its targets . Similarly, a kinase-dead mutant (K299M-Fyn) fails to phosphorylate target ITAMs and subsequently cannot establish stable interactions . These findings highlight the sequential and cooperative nature of p59-Fyn interactions: proper membrane localization through fatty acylation enables phosphorylation of target proteins by the kinase domain, creating binding sites for the SH2 domain.

What are the recommended approaches for studying p59-Fyn interactions with the T cell receptor?

When investigating p59-Fyn interactions with the TCR, several methodological considerations are crucial. First, cell lysis conditions significantly impact the detection of these interactions. Research indicates that mild detergents must be used to preserve the complex, as harsher conditions disrupt the association . Digitonin is often preferred over stronger detergents like Triton X-100 or NP-40.

For co-immunoprecipitation experiments, the following protocol has proven effective:

• Use COS-1 cells transfected with p59-Fyn constructs and CD8-ζ fusion proteins (e.g., 5 μg pEFBOS-CD8-ζ with 5 μg pCMV5-Fyn)

• Lyse cells in digitonin-containing buffer to preserve membrane integrity

• Immunoprecipitate complexes using anti-CD8 monoclonal antibodies

• Detect p59-Fyn in the precipitates using specific antibodies (e.g., anti-Fyn SH43 polyclonal antibodies)

For studying the role of fatty acylation, the following approaches are valuable:

• Use of site-directed mutagenesis to generate specific mutants (G2A, C3S, K7,9A)

• Co-expression with N-myristoyltransferase (NMT) to restore acylation in mutants

• Use of metabolic inhibitors like 2-hydroxymyristate to block myristoylation

• Generation of chimeric constructs (e.g., FynKRas) to study membrane targeting

What techniques are most effective for evaluating p59-Fyn enzymatic activity?

Evaluating p59-Fyn enzymatic activity requires specialized approaches to assess its tyrosine kinase function. The following methodologies are most effective:

• In vitro kinase assays:

  • Immunoprecipitate p59-Fyn from cell lysates

  • Incubate with exogenous substrates (e.g., enolase) and [γ-32P]ATP

  • Analyze phosphorylation by SDS-PAGE and autoradiography

  • Include appropriate controls: kinase-dead mutants (K299M-Fyn) and specific inhibitors

• Phosphotyrosine immunoblotting:

  • Transfect cells with wild-type or mutant p59-Fyn constructs

  • Lyse cells and perform immunoblotting with anti-phosphotyrosine antibodies

  • Measure changes in cellular protein tyrosine phosphorylation as an indication of kinase activity

• Fluorescence-based assays:

  • Utilize fluorescent peptide substrates containing optimal Fyn target sequences

  • Monitor phosphorylation through changes in fluorescence polarization or FRET

  • This approach allows for real-time, quantitative assessment of kinase activity

• Inhibition assays:

  • Test compounds for their ability to inhibit p59-Fyn kinase activity

  • Calculate IC50 values for potential inhibitors

  • Compare activity profiles against other Src-family kinases to assess specificity

When interpreting results, it's essential to consider that p59-Fyn activity may be influenced by multiple factors, including its phosphorylation state, membrane localization, and interactions with regulatory proteins. Therefore, complementary approaches should be used to comprehensively characterize its enzymatic properties.

What expression systems are optimal for producing recombinant p59-Fyn for structural and functional studies?

The choice of expression system for p59-Fyn production depends on the specific research objectives:

• Bacterial expression (E. coli):

  • Most commonly used for producing recombinant p59-Fyn domains (e.g., aa 23-216)

  • Advantages: High yield, cost-effective, straightforward purification (especially with tags)

  • Limitations: Lacks eukaryotic post-translational modifications, particularly lipid modifications

  • Optimal for structural studies of individual domains (SH2, SH3) and in vitro biochemical assays

  • Typically expressed with GST tags to enhance solubility and facilitate purification

• Mammalian expression systems:

  • COS-1 cells are frequently used for p59-Fyn expression in cellular studies

  • Advantages: Proper post-translational modifications (especially fatty acylation), correct folding

  • Suitable for studying interactions with other proteins and cellular localization

  • Transfection protocols typically use 2-5 μg of Fyn cDNA per experiment

  • For co-expression studies with NMT, ratios of 1:2 or 1:5 (Fyn:NMT) are effective

• Insect cell systems:

  • Baculovirus-infected Sf9 or High Five cells

  • Compromise between bacterial and mammalian systems

  • Higher yield than mammalian cells with many eukaryotic post-translational modifications

  • Suitable for producing full-length, active p59-Fyn for enzymatic studies

For purification, proprietary chromatographic techniques are typically employed . The choice of purification strategy should consider the presence of tags (e.g., GST) and the intended experimental application. When studying membrane-associated functions, it's crucial to preserve the native lipid modifications, making mammalian expression systems preferable despite their lower yield.

How can researchers address contradictory findings regarding p59-Fyn interactions with T cell receptor components?

Contradictory findings regarding p59-Fyn interactions with TCR components often arise from methodological differences and context-dependent behaviors. To address these discrepancies, consider the following approach:

• Critically evaluate experimental conditions:

  • Cell lysis conditions significantly impact detection of p59-Fyn/TCR interactions

  • Mild detergents (e.g., digitonin) preserve complexes that stronger detergents disrupt

  • The sensitivity of these interactions to detergent conditions suggests they occur in specialized membrane microdomains (lipid rafts)

• Consider the multifactorial nature of the interaction:

  • Initial studies identified residues G2, C3, K7, and K9 as an "ITAM recognition motif"

  • Later research revealed these residues actually ensure proper fatty acylation rather than directly mediating ITAM binding

  • Both findings are reconciled by understanding the sequential requirements: acylation → membrane targeting → kinase activity → SH2 binding

• Utilize multiple complementary approaches:

  • Combine biochemical (co-IP), genetic (mutagenesis), and pharmacological (acylation inhibitors) approaches

  • Employ both gain-of-function (NMT overexpression) and loss-of-function (mutants) strategies

  • Include appropriate controls for each experimental variable

When presenting and interpreting data, acknowledge the limitations of each experimental approach and consider how multiple mechanisms may contribute to the observed phenomena. The relationship between p59-Fyn and TCR components involves a sequence of events (fatty acylation, membrane targeting, kinase activity, and SH2-phosphotyrosine interactions), with disruption at any stage potentially yielding seemingly contradictory results.

What are the key considerations when designing experiments to study p59-Fyn in lipid rafts and membrane microdomains?

Studying p59-Fyn in lipid rafts requires specialized techniques to preserve and analyze these sensitive membrane microdomains:

• Isolation of detergent-resistant membranes (DRMs):

  • Lyse cells in cold 1% Triton X-100 buffer

  • Subject lysates to sucrose density gradient centrifugation

  • Collect and analyze fractions for p59-Fyn distribution

  • Include appropriate markers: glycosylphosphatidylinositol (GPI)-anchored proteins (positive raft markers) and transferrin receptor (negative raft marker)

• Microscopy approaches:

  • Fluorescence resonance energy transfer (FRET) between p59-Fyn and raft markers

  • Super-resolution microscopy techniques (STORM, PALM) to visualize nanoscale distribution

  • Use chimeric constructs (e.g., Fyn(16)-GFP) for live-cell imaging

• Manipulation of membrane rafts:

  • Cholesterol depletion using methyl-β-cyclodextrin

  • Comparison of wild-type Fyn with mutants having altered acylation (G2A, C3S)

  • Analysis of FynKRas chimera, which is farnesylated but excluded from rafts

• Controls and comparative analysis:

  • Include other Src-family kinases (e.g., Lck) with different membrane targeting properties

  • Use Lck(10)Fyn chimeras to study domain-specific contributions to localization

  • Compare multiple cell types, as raft composition varies between cells

Evidence indicates that p59-Fyn localization to membrane rafts is essential for its stable association with the TCR ζ chain . Experiments disrupting this localization (through altered acylation or raft disruption) consistently show reduced TCR interaction, highlighting the importance of considering membrane microdomain organization when studying p59-Fyn function.

How should researchers interpret differences in p59-Fyn isoform expression across different tissues?

Interpreting p59-Fyn isoform expression patterns requires careful consideration of several factors:

• Quantitative analysis methods:

  • RT-PCR to detect isoform-specific mRNA levels

  • Western blotting with isoform-specific antibodies

  • Mass spectrometry for unbiased protein identification and quantification

  • RNA sequencing to identify novel splice variants

• Tissue-specific expression patterns:

  • p59-Fyn is highly expressed in brain tissue, suggesting specialized roles in neural cells

  • Distinct isoforms exist due to alternative splicing, with tissue-specific distribution

  • When comparing across tissues, normalize expression to appropriate housekeeping genes/proteins

• Functional implications:

  • Correlate isoform expression with known tissue-specific functions

  • In brain tissue, p59-Fyn may contribute to myelination during CNS development

  • In T cells, p59-Fyn interacts with TCR components to regulate immune responses

  • Different isoforms may have distinct substrate preferences or regulatory mechanisms

• Developmental considerations:

  • Expression patterns may change during development

  • Early CNS formation involves p59-Fyn in myelination processes

  • Temporal expression analysis across developmental stages provides insight into changing functions

What are the emerging approaches for targeting p59-Fyn in neurological and immunological disorders?

Research into p59-Fyn targeting strategies is advancing in both neurological and immunological contexts:

• Neurological applications:

  • p59-Fyn's high expression in brain and role in myelination make it a target in neurological disorders

  • Inhibitors targeting p59-Fyn kinase activity are being explored for demyelinating diseases

  • Selective modulation of brain-specific isoforms offers potential for reduced off-target effects

  • Approaches targeting p59-Fyn/tau interactions show promise in Alzheimer's disease models

• Immunological applications:

  • p59-Fyn's role in T cell signaling makes it relevant in autoimmune conditions and transplant rejection

  • Selective inhibition of p59-Fyn activity may modulate T cell responses without complete immunosuppression

  • Targeting the specific interaction between p59-Fyn and TCR components could provide precise immunomodulation

• Novel targeting strategies:

  • Lipid raft-targeted delivery systems to concentrate therapeutics where p59-Fyn is active

  • Peptide-based inhibitors mimicking specific protein-protein interaction domains

  • Allosteric modulators targeting regulatory interactions rather than catalytic activity

  • Dual fatty acylation inhibitors to prevent membrane localization and function

• Methodological considerations:

  • Use of conditional knockout models to assess tissue-specific functions

  • Development of isoform-selective inhibitors based on structural differences

  • High-throughput screening platforms incorporating membrane context

  • Advanced imaging techniques to monitor drug effects on p59-Fyn localization and interactions

Future research should focus on developing more selective approaches that target specific functions of p59-Fyn while minimizing effects on related kinases. Additionally, understanding the context-dependent roles of p59-Fyn in different tissues will be essential for developing targeted therapeutic strategies with minimal side effects.

What computational approaches are most effective for predicting p59-Fyn interaction networks and identifying potential substrates?

Computational prediction of p59-Fyn interactions and substrates requires sophisticated approaches:

• Sequence-based prediction methods:

  • Scansite and similar tools to identify potential phosphorylation sites based on consensus motifs

  • SH2/SH3 domain binding site prediction using position-specific scoring matrices

  • Machine learning algorithms trained on known Src-family kinase substrates

  • Sequence conservation analysis across species to identify functionally important sites

• Structural modeling approaches:

  • Molecular docking to predict interactions between p59-Fyn domains and potential partners

  • Molecular dynamics simulations to assess stability of predicted complexes

  • Assessment of binding free energy to rank potential interactions

  • Integration of structural information with experimental data (e.g., hydrogen-deuterium exchange)

• Network-based approaches:

  • Protein-protein interaction databases to identify known p59-Fyn interactors

  • Pathway enrichment analysis to identify biological processes involving p59-Fyn

  • Network analysis to identify key nodes that may represent critical substrates

  • Integration of phosphoproteomic data with interaction networks

• Validation strategies:

  • in vitro kinase assays with predicted substrates

  • Co-immunoprecipitation of predicted interaction partners

  • Mutational analysis of predicted binding sites

  • Correlation of computational predictions with biological phenotypes

The most effective approach combines multiple computational methods with experimental validation in an iterative process. Initial predictions guide focused experiments, with results feeding back to refine computational models. This integrated approach has successfully identified novel p59-Fyn substrates and interaction partners in various cellular contexts.

Key Structural Features of p59-Fyn and Their Functional Significance

Comparison of Experimental Approaches for Studying p59-Fyn Function