Recombinant Tyrosine-protein kinase transforming protein Abl (ABL), partial

What is the domain organization of ABL kinases and how does it relate to their function?

ABL-family proteins comprise one of the best conserved branches of tyrosine kinases. Each ABL protein contains an SH3-SH2-TK (Src homology 3–Src homology 2–tyrosine kinase) domain cassette, which confers autoregulated kinase activity and is common among nonreceptor tyrosine kinases . This cassette is coupled to an actin-binding and bundling domain, enabling ABL proteins to connect phosphoregulation with actin-filament reorganization .

Two vertebrate paralogs, ABL1 and ABL2, have evolved to perform specialized functions:

• ABL1 includes nuclear localization signals and a DNA binding domain that mediates DNA damage-repair functions

• ABL2 has additional binding capacity for actin and microtubules to enhance cytoskeletal remodeling functions

This structural organization makes ABL kinases unique signaling molecules capable of integrating cytoskeletal dynamics with phosphorylation cascades.

What mechanisms regulate ABL kinase activity?

Multiple autoinhibitory mechanisms constrain the enzymatic activity of ABL-family kinases:

• Myristoylation: A myristoyl group attached to the amino-terminal glycine of ABL1 and ABL2 "b" isoform proteins nestles into a surface pocket in the kinase domain, contributing to an autoinhibitory fold .

• N-terminal "Cap": A short amino-terminal sequence stabilizes the inactive conformation through additional surface interactions .

• SH3-SH2 domain interactions: These domains cradle the kinase domain, imposing a "locked" inactive state .

• Phosphatidylinositol binding: PIP₂ binds the ABL1 SH2 domain through residues normally required for phosphotyrosine binding, although the precise inhibition mechanism remains undetermined .

• Tyrosine phosphorylation: Key phosphorylation events can either activate or inhibit the kinase:

  • Phosphorylation of ABL1-Y245 (ABL2-Y272) and ABL1-Y412 (ABL2-Y439) correlates with increased kinase activity

  • Phosphorylation of ABL1-Y89 disrupts SH3 domain-based autoinhibitory interactions and enhances activity

  • Phosphorylation of ABL1-Y272 inhibits activity, while phosphorylation of nearby ABL1-Y276 enhances activity

• Allosteric SH2-kinase interactions: These interactions switch the Abl activation loop from a closed to a fully open conformation, enabling trans-autophosphorylation of the activation loop .

What expression systems are most effective for producing active recombinant ABL kinases?

While baculovirus-infected insect cells have traditionally been used for large-scale ABL expression, bacterial systems can be effectively optimized for ABL production:

Bacterial Expression (E. coli):

• MBP fusion strategy: Expressing ABL as a Maltose Binding Protein (MBP) fusion dramatically increases solubility, with >90% of the recombinant fusion remaining in soluble cell lysate after centrifugation .

• Co-expression with phosphatases: Co-expression with YopH phosphatase allows purification in the dephosphorylated state .

• Stabilization with inhibitors: Adding inhibitors such as PD166326 or imatinib (depending on phosphorylation status) significantly improves protein stability .

• His-tag purification: His₆-tag/Co²⁺ affinity purification is more efficient than MBP/amylose for isolation .

Yield comparison:
Bacterial expression can yield several mg/L of soluble protein in minimal media , which is sufficient for most structural and biochemical studies.

Notably, the ABL kinase domain expressed in bacterial cells as an MBP fusion adopts the same folding as proteins expressed in higher eukaryotic cells, as confirmed by NMR spectroscopy . This makes bacterial expression a cost-effective alternative to insect cell systems for many applications.

How can phosphorylation status be controlled in recombinant ABL production?

Controlling phosphorylation status is critical for ABL functionality studies:

• For dephosphorylated ABL:

  • Co-expression with phosphatases (like YopH) during production

  • Post-purification treatment with calf intestinal alkaline phosphatase (CIP) at 20 units/ml at 20°C

• For phosphorylated ABL:

  • Leverage auto-phosphorylation capacity - mass spectrometry analysis of bacterially-expressed ABL revealed phosphorylation at multiple tyrosine residues (Y272, Y276, Y283, Y412, and Y468), including the key activation loop site Y412

  • When expressed in E. coli without phosphatase co-expression, recombinant ABL auto-phosphorylates and activates itself, further phosphorylating other bacterial proteins

• Site-specific phosphorylation control:

  • Mutations at key auto-phosphorylation sites can be introduced to prevent or mimic phosphorylation

  • Point mutations (e.g., K7A, W118A, E157A, Y158D, or the P242E/P249E double mutation) can disrupt autoinhibitory interactions and increase kinase activity

The phosphorylation state dramatically affects inhibitor binding preferences, with dephosphorylated ABL preferentially binding to type II inhibitors like imatinib, while phosphorylated ABL preferentially binds type I inhibitors like PD166326 .

What are reliable methods for measuring ABL kinase activity?

Several well-established methods can quantify ABL kinase activity:

1. FRET-based Z'Lyte kinase assay:

• Uses a peptide substrate (Tyr2) tagged with coumarin and fluorescein, forming a FRET pair

• After phosphorylation by ABL, a site-specific protease cleaves only unphosphorylated peptide, causing loss of FRET signal

• Procedure:

  • Titrate ABL protein (0.2–200 ng/well) in assay buffer

  • Add ATP (50 μM) plus Tyr2 peptide substrate (1 μM)

  • Incubate for 1 hour

  • Add development protease

  • Measure coumarin and fluorescein fluorescence

  • Calculate percent phosphorylation relative to control peptides

2. ADP-Glo™ kinase assay:

• Measures ADP production during the kinase reaction

• Can be used with various peptide substrates, including Abltide

3. Phosphate release assay using malachite green:

• Detects released inorganic phosphate from the kinase reaction

• Calculation of specific activity:
  $Specific Activity (pmol/min/μg) = \frac{Phosphate released (nmol) × 1000 pmol/nmol}{Incubation time (min) × amount of enzyme (μg)}$

4. Bacterial co-expression system:

• Co-express ABL kinase with a substrate peptide (e.g., Abltide) tagged with GST in E. coli

• Detect tyrosine phosphorylation of the substrate using Phos-tag SDS-PAGE, followed by western blotting

• Can be used to compare relative activities of wild-type versus mutant ABL proteins

5. Western blot for phosphorylation of endogenous substrates:

• Direct, same-lane comparison of tyrosine-phosphorylated and non-phosphorylated levels of the direct BCR-ABL substrate CrkL

• FACS-based readouts of BCR-ABL tyrosine phosphorylation substrates (pSTAT5 and pCrkL)

How can different ABL mutations be tested for kinase activity and inhibitor response?

Testing ABL mutations is crucial for understanding resistance mechanisms and structure-function relationships:

Experimental approach for comparing ABL mutant activities:

• Expression and purification:

  • Express wild-type and mutant ABL proteins using identical conditions

  • Verify protein purity and mass by SDS-PAGE and mass spectrometry

• Activity comparison methods:

  • In vitro kinase assays: Compare kinetic parameters (Kₘ and Vₘₐₓ) using optimal Abl substrate peptides

  • Enzyme-kinetic experiments: The Bcr-Abl I164E mutant protein, for example, displayed a >3-fold reduction in Vₘₐₓ compared to wild-type and a modest increase in Kₘ

  • Bacterial co-expression system: Used to compare wild-type ABL with five kinase domain mutants (Y253F, E255K, T315I, M351T, and H396P) associated with imatinib resistance

• Inhibitor response testing:

  • Transient exposure studies: Expose cells expressing different ABL mutants to inhibitors, then measure biochemical parameters and apoptosis

  • Dissociation kinetics: Measure inhibitor binding and dissociation rates using TR-FRET

Research findings from mutant studies:

• Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants, and undetectable in T315I and M351T mutants

• The SH2-kinase interface mutation I164E led to strong reduction of Abl autophosphorylation and in vitro kinase activity

• Mutation of Ile164 to different polar/charged amino acids (Glu, Gln, Thr, Asp, or Lys) or to Ala led to strong reduction of activity, while mutation to structurally related hydrophobic amino acids (Val or Leu) had somewhat weaker effects

How can fractional factorial design be applied to optimize experimental conditions for ABL kinase assays?

Fractional factorial design (FFD) offers an efficient experimental approach for optimizing ABL kinase assays when multiple factors affect enzyme activity:

Principles for applying FFD to ABL kinase experiments:

• Identify key experimental factors:

  • Buffer components (pH, salt concentration, divalent cations)

  • Reaction conditions (temperature, time, enzyme concentration)

  • Substrate characteristics (concentration, type)

  • Additives (stabilizers, detergents, reducing agents)

• Design the experiment:

  • Use a 2ᵏ⁻ᵖ fractional factorial design where k is the number of factors and p determines the fraction (1/2ᵖ) of the full factorial design to be run

  • For example, with 5 factors at 2 levels each, a 2⁵⁻¹ design would require 16 runs instead of 32

  • Select high (+1) and low (-1) levels for each factor based on literature or preliminary data

• Analyze results with aliasing patterns in mind:

  • Main effects may be confounded with higher-order interactions

  • Analysis should consider aliased effects

• Resolve ambiguities:

  • Follow the hierarchical ordering principle (lower-order effects are more likely important)

  • Use the effect sparsity principle (relatively few effects are significant)

  • Consider sequential experiments or fold-over designs for further clarification

This approach can significantly reduce the number of experiments needed to optimize ABL kinase assay conditions while still providing robust statistical analysis of main effects.

How can SH2-kinase domain interactions be evaluated experimentally?

The allosteric interactions between the SH2 domain and kinase domain are critical for ABL regulation. These can be evaluated through several experimental approaches:

1. FKBP-FRB fusion protein system:

• Express SH2 and kinase domains of Abl separately

• Induce their physical association upon addition of rapamycin

• Measure the resulting phosphorylation of cotransfected substrates (e.g., paxillin) and total cellular tyrosine phosphorylation

• Use I164E mutation to abolish the SH2-kinase interface interaction as a control

2. X-ray scattering solution structures:

• Analyze multidomain c-Abl kinase core proteins modeling diverse active states

• Compare wild-type with activating mutations like A356N (myristic acid binding pocket mutant)

• Results have shown that activation can occur with or without global allosteric changes in the core structure

3. Point mutations in the SH2-kinase interface:

• Mutations in the SH2 domain (e.g., I164E) disrupt the SH2-kinase interface and abolish activation loop phosphorylation

• Mutation of Ile164 to structurally related hydrophobic amino acids (Val or Leu) has weaker effects than mutation to polar/charged residues

4. SH2 domain replacement experiments:

• Replace the ABL SH2 domain with SH2 domains from other proteins

• Observe the shift in substrate profile, which correlates with SH2 binding preference

• This demonstrates the SH2 domain's contribution to catalytic activity and target site specificity

Research has shown that these allosteric SH2-kinase domain interactions switch the Abl activation loop from a closed to a fully open conformation, enabling trans-autophosphorylation of the activation loop and requiring prior phosphorylation of the SH2-kinase linker .

How does ABL kinase function in cancer progression and metastasis?

ABL kinases demonstrate context-dependent roles in cancer progression:

Tumor-promoting functions:

• Constitutively active BCR-ABL1 fusion protein drives chronic myeloid leukemia (CML)

• In many solid tumors, Abl kinases are regarded as promoters of tumor progression and metastasis

Tumor-suppressive functions:

• ABL1 can mediate pro-apoptotic functions in response to DNA damage

• Recent studies show Abl kinases can restrain malignant behavior in multiple settings

• Loss of ABL and ARG in prostate cancer models leads to:

  • Significantly increased tumor burdens (over an order of magnitude greater than control tumors)

  • Enhanced dissemination to kidney, liver, and lung

  • Dramatically enhanced 3D growth on soft collagen matrix

Molecular mechanisms:

• ABL/ARG knockdown cells show sustained AKT signaling, potentially explaining enhanced tumor growth

• The effect is context-specific, as differences are largely attenuated under standard tissue culture conditions

These findings reveal that Abl family kinases can function as suppressors of tumor progression and metastasis in some contexts, contrary to their well-established role in promoting leukemogenesis through BCR-ABL fusion proteins.

What experimental approaches can determine threshold levels of ABL inhibitors required for biological effects?

Understanding the relationship between ABL inhibitor retention and cellular response is critical for therapeutic development:

Experimental framework:

• Transient exposure studies:

  • Expose CML cells transiently to ABL tyrosine kinase inhibitors (TKIs)

  • Measure multiple parameters after drug washout:

    • Intracellular drug concentration (LC/MS/MS)

    • BCR-ABL signaling (immunoblot, FACS)

    • Apoptosis markers

    • ABL-inhibitor dissociation kinetics

• Biochemical dissociation studies:

  • Use tracer binding and TR-FRET signal measurements

  • Establish dissociation curves to determine off-rate (t₁/₂) values

• Signal restoration assessment:

  • Monitor restoration of BCR-ABL signaling using:

    • Immunoblot assay comparing tyrosine-phosphorylated and non-phosphorylated levels of the direct BCR-ABL substrate CrkL

    • FACS-based readouts of BCR-ABL phosphorylation substrates (pSTAT5 and pCrkL)

Key findings:

• Attenuated restoration of BCR-ABL signaling correlates with apoptosis commitment

• Intracellular retention of ABL TKIs above a quantifiable threshold is a critical parameter mediating this effect

• This understanding of threshold levels can inform dosing strategies and development of next-generation inhibitors

What are common issues in recombinant ABL expression and how can they be resolved?

Several challenges may arise during recombinant ABL production, with specific solutions:

How can segmental isotopic labeling be achieved for NMR studies of ABL kinase?

For detailed structural studies of ABL kinase domains using NMR spectroscopy, segmental isotopic labeling provides significant advantages:

Methodology for segmental isotopic labeling of ABL:

• Express protein fragments separately:

  • Express different domains or segments of ABL kinase with appropriate fusion tags

  • Ensure one segment is isotopically labeled (¹⁵N, ¹³C) by growing in minimal media with labeled nutrients

• Use expressed protein ligation (EPL):

  • Bacterial expressed Abl kinase domain constructs can achieve segmental isotopic labeling using EPL

  • For C-terminal segments, express as intein fusions to generate thioesters

  • For N-terminal segments, include a protease site or chemical cleavage site to expose the required N-terminal residue

• Perform ligation reaction:

  • Mix the purified protein segments under appropriate buffer conditions

  • Add catalysts if needed for the specific ligation chemistry

  • Purify the ligated product using affinity chromatography and size exclusion

• Verify intact structure:

  • Compare HSQC/TROSY spectra for the segmentally labeled ABL kinase with spectra from uniformly labeled proteins

  • Bacterial expressed ABL kinase shows similar corresponding chemical shifts to ABL expressed in baculovirus-infected insect cells with either amino-acid-type selective labeling or uniform labeling

This approach is especially useful for NMR structure/activity studies of larger multi-domain ABL constructs, allowing researchers to focus on specific domains while reducing spectral complexity.