GUK1 Antibody

What is GUK1 and what are its fundamental biological functions?

GUK1 (guanylate kinase 1) is a 197 amino acid protein characterized by a guanylate kinase-like domain and belongs to the guanylate kinase family. It functions primarily as a metabolic enzyme that catalyzes the ATP-dependent conversion of guanosine monophosphate (GMP) to guanosine diphosphate (GDP), playing a vital role in GMP recycling . This recycling process is essential for maintaining the balance of guanine nucleotides, which are critical components of various signal transduction pathways. GUK1 is ubiquitously expressed in tissues and functions as a monomer, indicating its fundamental role in cellular metabolism . The protein has a calculated molecular weight of 22 kDa but is typically observed at 22-27 kDa in experimental conditions .

What applications are GUK1 antibodies validated for in research settings?

GUK1 antibodies have been validated for multiple research applications:

Researchers should note that optimal dilutions may be sample-dependent, and it is recommended to titrate the antibody in each testing system to obtain optimal results .

What species reactivity has been confirmed for commonly used GUK1 antibodies?

Most commercial GUK1 antibodies, including the monoclonal antibody products (67047-1-Ig and C-4), have been validated for reactivity with human, mouse, and rat samples . The cross-species reactivity makes these antibodies valuable tools for comparative studies across model organisms. This broad reactivity profile is particularly useful for translational research that bridges findings from animal models to human applications, especially in cancer research where GUK1's role is increasingly recognized.

What is the recommended protocol for antigen retrieval when using GUK1 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of GUK1, the primary recommended protocol involves antigen retrieval with TE buffer at pH 9.0 . This method has been validated on multiple tissue types including human thyroid cancer tissue and human prostate cancer tissue. Alternatively, when working with tissues that show suboptimal results with the primary method, citrate buffer at pH 6.0 can be used as an alternative antigen retrieval approach . Complete protocols typically include:

• Deparaffinization and rehydration of tissue sections

• Antigen retrieval using TE buffer (pH 9.0) in a pressure cooker or water bath

• Blocking of endogenous peroxidase activity

• Application of primary GUK1 antibody at 1:400-1:1600 dilution

• Detection with appropriate secondary antibody and visualization system

• Counterstaining, dehydration, and mounting

Always verify specific protocol details for your particular antibody from the manufacturer's guidelines.

How should GUK1 antibodies be stored to maintain optimal reactivity?

GUK1 antibodies should be stored at -20°C in appropriate buffer conditions to maintain their reactivity and specificity. For example, the 67047-1-Ig antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage of this formulation. Some suppliers provide small volume formats (20μl) that contain 0.1% BSA for additional stability . It's important to minimize freeze-thaw cycles to preserve antibody functionality. Always refer to the specific storage conditions recommended by the manufacturer of your particular GUK1 antibody preparation.

What is the emerging role of GUK1 in cancer metabolism, particularly in lung cancers?

Recent research published in February 2025 has identified GUK1 as a critical metabolic enzyme that promotes growth in certain lung cancers, particularly those harboring alterations in the ALK gene . GUK1 supports cancer cell metabolism by enhancing the synthesis of GDP, which is a precursor to the energy-rich molecule GTP that cancer cells require for various processes including cell division and protein synthesis .

The metabolic dependency on GUK1 appears to be particularly pronounced in ALK-positive lung cancers. When researchers disabled GUK1 in experimental models, cancer cell growth slowed considerably, indicating that these cancers become highly dependent on this enzyme as a metabolic vulnerability . Additionally, evidence suggests elevated GUK1 levels in multiple subtypes of lung cancer beyond those with ALK alterations, pointing to a potentially broader role of this enzyme in lung cancer metabolism .

The identification of GUK1 as a metabolic dependency represents a significant advance in understanding the molecular underpinnings of lung cancer metabolism and highlights GUK1 as a potential therapeutic target for future development .

What is the molecular mechanism by which ALK fusion proteins interact with GUK1 in cancer cells?

The molecular interaction between ALK fusion proteins and GUK1 involves a phosphorylation mechanism that enhances metabolic activity. Research published in Cell (February 2025) demonstrated that in lung cancers with ALK gene alterations, the abnormal ALK protein specifically phosphorylates GUK1 at tyrosine 74 . This post-translational modification enhances GUK1's enzymatic activity, resulting in increased GDP biosynthesis .

The phosphorylation of GUK1 creates a metabolic cascade where:

• Activated ALK phosphorylates GUK1 at Y74

• Phosphorylated GUK1 exhibits enhanced catalytic efficiency

• Increased GMP to GDP conversion occurs

• Elevated GDP levels support increased GTP synthesis

• Higher GTP availability fuels cancer cell division and protein synthesis

This mechanism reveals how oncogenic fusion kinases like ALK can hijack normal metabolic enzymes to support cancer growth. Interestingly, evidence suggests that other oncogenic fusion kinases may converge on this same pathway, explaining why GUK1 appears to be relevant in multiple subtypes of lung cancer . This mechanistic insight provides a foundation for developing targeted approaches to disrupt this cancer-promoting metabolic pathway.

How can researchers effectively evaluate GUK1 expression levels in tumor samples?

Researchers can employ multiple complementary approaches to accurately assess GUK1 expression levels in tumor samples:

Immunohistochemistry (IHC):

• Use validated anti-GUK1 antibodies at recommended dilutions (1:400-1:1600)

• Follow antigen retrieval protocols with TE buffer (pH 9.0)

• Include appropriate positive controls (thyroid cancer tissue, prostate cancer tissue)

• Quantify expression using standard scoring systems (H-score or percentage positive cells)

Western Blot Analysis:

• Extract proteins using detergent-based lysis buffers

• Load 20-40 μg total protein per lane

• Use validated GUK1 antibodies at 1:1000-1:5000 dilution

• Expect bands at 22-27 kDa

• Use appropriate loading controls (β-actin, GAPDH) for normalization

Quantitative RT-PCR:

• Design primers specific to GUK1 mRNA

• Normalize expression to suitable housekeeping genes

• Compare expression across samples using the 2^-ΔΔCT method

Mass Spectrometry-Based Proteomics:

• For unbiased detection and quantification of GUK1 and post-translational modifications

• Can provide absolute quantification when using labeled standards

When evaluating GUK1 expression, researchers should consider measuring both total GUK1 levels and phosphorylated GUK1 (particularly at Y74) to fully understand the activation state of this pathway in tumor samples .

What experimental models are most appropriate for studying GUK1 function in lung cancer?

Based on recent research findings, several experimental models have proven effective for studying GUK1 function in lung cancer:

Cell Line Models:

• ALK-positive lung cancer cell lines (e.g., H3122, H2228)

• Cell lines with experimentally induced ALK alterations

• HEK-293 and HeLa cells for basic mechanistic studies

Patient-Derived Models:

• Patient-derived xenografts (PDXs) from ALK-positive lung cancers

• Primary patient-derived cell cultures that maintain ALK signaling

Genetic Modification Approaches:

• CRISPR/Cas9-mediated GUK1 knockout or knockdown models

• Site-directed mutagenesis of the Y74 phosphorylation site

• Inducible shRNA or siRNA systems for temporal control of GUK1 expression

In Vivo Mouse Models:

• Genetically engineered mouse models (GEMMs) with ALK alterations

• Orthotopic lung cancer models with modulated GUK1 expression

When designing experiments, researchers should consider including both gain-of-function (GUK1 overexpression) and loss-of-function (GUK1 knockdown) approaches to comprehensively understand GUK1's role. Additionally, phosphomimetic mutants (Y74D or Y74E) and phospho-dead mutants (Y74F) can provide valuable insights into the specific effects of GUK1 phosphorylation on cancer cell metabolism and growth .

What are the technical considerations for developing selective inhibitors targeting GUK1?

Developing selective inhibitors targeting GUK1 presents several technical considerations that researchers must address:

Structural Basis for Selectivity:

• Crystal structures of GUK1 (both unphosphorylated and phosphorylated at Y74) would be essential for structure-based drug design

• Understanding the conformational changes induced by ALK-mediated phosphorylation

• Identifying unique binding pockets that distinguish GUK1 from other guanylate kinases

Target Site Selection:

• ATP-competitive inhibitors (targeting the ATP-binding site)

• GMP-competitive inhibitors (targeting the substrate-binding site)

• Allosteric inhibitors (targeting regulatory sites affected by Y74 phosphorylation)

• Protein-protein interaction inhibitors (disrupting ALK-GUK1 binding)

Assay Development:

• Biochemical assays measuring GUK1 enzymatic activity (GMP to GDP conversion)

• Cellular assays monitoring GDP/GTP levels in cancer cells

• Phosphorylation-specific assays for Y74 phosphorylation

• Cell viability assays in ALK-positive versus ALK-negative cells

Selectivity Profiling:

• Cross-screening against other guanylate kinases and nucleotide kinases

• Testing effects on normal cells versus cancer cells

• Evaluation of on-target versus off-target effects in vivo

Pharmacological Considerations:

• Physicochemical properties suitable for cell permeability

• Metabolic stability appropriate for in vivo testing

• Delivery strategies to achieve sufficient concentration in lung tissue

The recent identification of GUK1 as a metabolic vulnerability in lung cancer suggests that successful development of selective GUK1 inhibitors could provide a novel therapeutic approach for patients with ALK-positive lung cancers and potentially other lung cancer subtypes with elevated GUK1 dependency.

How can researchers distinguish between specific GUK1 effects and compensatory metabolic pathways in experimental systems?

Distinguishing specific GUK1 effects from compensatory metabolic adaptations requires careful experimental design and multiple complementary approaches:

Temporal Analysis:

• Use inducible knockdown/knockout systems for GUK1

• Monitor metabolic changes at multiple timepoints after GUK1 inhibition

• Identify immediate effects (likely direct) versus delayed effects (possibly compensatory)

Metabolic Flux Analysis:

• Employ stable isotope tracing (e.g., 13C-labeled glucose or glutamine)

• Track the fate of labeled atoms through guanine nucleotide pathways

• Measure changes in flux through GUK1-dependent and alternative pathways

Comprehensive Metabolomics:

• Perform untargeted metabolomics to identify unexpected metabolic adaptations

• Quantify changes in GDP/GTP and related nucleotides

• Measure potential accumulation of upstream metabolites (GMP) and depletion of downstream products

Rescue Experiments:

• Test whether exogenous GDP or GTP supplementation can rescue phenotypes

• Express GUK1 mutants resistant to your inhibition strategy to confirm specificity

• Introduce alternative enzymes that can bypass the GUK1-dependent step

Multi-omics Integration:

• Combine metabolomics with transcriptomics to identify upregulated compensatory enzymes

• Use proteomics to detect changes in expression or post-translational modifications of related metabolic enzymes

• Employ computational modeling to predict metabolic rewiring after GUK1 inhibition

By systematically implementing these approaches, researchers can differentiate between direct GUK1-dependent effects and secondary adaptations that emerge as cancer cells attempt to maintain nucleotide homeostasis after GUK1 inhibition. This distinction is crucial for understanding potential resistance mechanisms that might emerge during therapeutic targeting of GUK1 in lung cancers .

What are the optimal controls and validation methods when using GUK1 antibodies in cancer research studies?

When using GUK1 antibodies for cancer research, implementing proper controls and validation methods is essential for generating reliable and reproducible results:

Antibody Validation Controls:

Method-Specific Validation:

For Western blot:

• Include molecular weight markers to confirm the expected 22-27 kDa band

• Use positive control lysates from validated cell lines

• Include loading controls for normalization

• Confirm knockdown efficiency when performing functional studies

For Immunohistochemistry/Immunofluorescence:

• Include tissue microarrays with known positive and negative samples

• Use antigen retrieval optimization (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Incorporate isotype control antibodies (Mouse IgG1 for clones like 67047-1-Ig)

• Perform dual staining with other markers to confirm cell type-specific expression

For functional studies:

• Compare multiple GUK1 inhibition approaches (siRNA, shRNA, CRISPR, small molecules)

• Rescue experiments with wild-type GUK1 expression

• Include phosphorylation-site mutants (Y74F) to validate ALK-GUK1 signaling

• Measure both protein levels and enzymatic activity

Implementing these rigorous controls and validation methods ensures that findings regarding GUK1's role in cancer metabolism and growth are robust and reproducible across different experimental systems and laboratory settings .

How does GUK1 expression correlate with clinical outcomes in lung cancer patients?

Recent research indicates that GUK1 expression levels may have significant correlations with clinical outcomes in lung cancer patients, particularly those with ALK-positive tumors. While comprehensive clinical correlation studies are still emerging, preliminary findings suggest:

ALK-Positive Lung Cancers:

• Higher GUK1 expression and particularly increased Y74 phosphorylation is observed in ALK-positive lung cancers

• GUK1 dependency appears to be especially pronounced in this molecular subtype, suggesting potential prognostic relevance

• ALK inhibitor resistance mechanisms may involve altered GUK1 regulation or bypass pathways

Broader Implications in Lung Cancer:

• Evidence suggests elevated GUK1 expression in additional subtypes of lung cancer beyond those with ALK alterations

• This indicates a potentially broader role of GUK1 in lung cancer metabolism and progression

• Multiple oncogenic fusion kinases appear to converge on GUK1 to augment GTP synthesis and promote lung cancer growth

Clinical Research Directions:

Researchers investigating clinical correlations should consider analyzing both total GUK1 expression and phospho-Y74 GUK1 levels in patient samples, as the phosphorylation status appears to be functionally significant in activating GUK1's oncogenic potential . Additionally, integrating GUK1 analysis with comprehensive molecular profiling of tumors may reveal important co-dependencies or synthetic lethal relationships that could inform personalized treatment approaches for lung cancer patients .

What are the most promising future research directions involving GUK1 antibodies in cancer research?

Several high-priority research directions involving GUK1 antibodies in cancer research have emerged from recent findings:

• Development of phospho-specific antibodies: Creating and validating antibodies that specifically recognize GUK1 phosphorylated at Y74 would enable more precise monitoring of ALK-mediated GUK1 activation in patient samples .

• Therapeutic targeting validation: Using GUK1 antibodies to monitor response to experimental GUK1 inhibitors or to validate target engagement in preclinical models.

• Biomarker development: Evaluating whether GUK1 expression or phosphorylation status, as detected by specific antibodies, can serve as predictive biomarkers for response to therapies targeting this metabolic pathway.

• Expansion to other cancer types: Investigating GUK1 expression and function beyond lung cancer, particularly in other malignancies where nucleotide metabolism plays a critical role.

• Single-cell analysis: Applying GUK1 antibodies in single-cell proteomic approaches to understand heterogeneity in GUK1 expression and activation within tumors.

These research directions build upon the identification of GUK1 as a key metabolic node in lung cancer and will potentially expand our understanding of how metabolic dependencies can be leveraged for cancer therapy development .

What interdisciplinary approaches might accelerate GUK1-focused cancer research?

Advancing GUK1-focused cancer research will benefit from integrating multiple scientific disciplines:

• Structural biology and medicinal chemistry: Determining the three-dimensional structure of GUK1, particularly in its phosphorylated state, to facilitate structure-based drug design of selective inhibitors.

• Systems biology and computational modeling: Developing comprehensive models of nucleotide metabolism to predict the consequences of GUK1 inhibition and identify potential resistance mechanisms.

• Chemical biology: Creating activity-based probes and chemoproteomics approaches to study GUK1 activity in complex cellular environments.

• Immunology and immuno-oncology: Investigating whether GUK1 inhibition affects cancer cell interactions with the immune microenvironment, potentially enhancing immunotherapy approaches.

• Translational research: Establishing patient-derived models that maintain GUK1 dependency to bridge laboratory findings to clinical applications.

• Clinical biomarker development: Creating standardized assays using GUK1 antibodies that can be implemented in clinical trials to stratify patients.