Recombinant Human Uncharacterized aarF domain-containing protein kinase 2 (ADCK2)
Description
Molecular Structure and Characteristics
ADCK2 belongs to the family of aarF domain-containing kinases, a subset of atypical protein kinases involved in mitochondrial functions. The recombinant form of human ADCK2 has a molecular weight of approximately 28.7 kDa when expressed for the region spanning amino acids 128-389 . The protein is typically produced with an N-terminal His Tag to facilitate purification and downstream applications . ADCK2 is also known by several alternative names including AARF, aarF domain containing kinase 2, MGC20727, and Putative ubiquinone biosynthesis protein AarF . As a protein kinase, ADCK2 functions by transferring phosphate groups from ATP to substrate molecules, thereby regulating various cellular signaling cascades .
Cellular Localization and Expression Patterns
ADCK2 is primarily localized within mitochondria, where it participates in essential metabolic processes . The protein plays a critical role in promoting the transport of lipids into mitochondria and is essential for mitochondrial fatty acid β-oxidation and Coenzyme Q (CoQ) biosynthesis . Analysis of ADCK2 expression across human tissues reveals variable distribution patterns, suggesting tissue-specific functions of this protein . This differential expression may contribute to the context-dependent roles of ADCK2 observed in various physiological and pathological conditions.
Role in Mitochondrial Metabolism
ADCK2 serves as a key regulator of mitochondrial function. Studies have demonstrated that depletion of ADCK2 in cancer cells disrupts normal mitochondrial processes, leading to cytochrome C release, mitochondrial depolarization, DNA damage, and ATP reduction . These findings highlight ADCK2's integral role in maintaining mitochondrial integrity and energy homeostasis. The mitochondrial dysfunction resulting from ADCK2 depletion can trigger apoptotic pathways and significantly impair cellular viability, particularly in cancer cells that rely heavily on mitochondrial metabolism for survival and proliferation.
Involvement in Lipid Transport and Fatty Acid Oxidation
One of ADCK2's primary functions involves facilitating lipid transport into mitochondria . This process is critical for fatty acid β-oxidation, a metabolic pathway that generates acetyl-CoA molecules for entry into the tricarboxylic acid (TCA) cycle and subsequent ATP production. By regulating lipid metabolism in mitochondria, ADCK2 contributes significantly to cellular energy production and metabolic homeostasis. Disruptions in ADCK2 function can therefore lead to alterations in lipid metabolism and energy production, potentially contributing to metabolic disorders and cancer pathogenesis.
Contribution to Coenzyme Q Biosynthesis
ADCK2 plays an essential role in the biosynthesis of Coenzyme Q (CoQ), a critical component of the electron transport chain in mitochondria . CoQ functions as an electron carrier in the inner mitochondrial membrane, facilitating ATP production through oxidative phosphorylation. Through its involvement in CoQ biosynthesis, ADCK2 indirectly contributes to cellular energy production and protection against oxidative stress. This function further emphasizes ADCK2's importance in maintaining mitochondrial function and cellular energy homeostasis.
ADCK2 in Non-Small Cell Lung Cancer (NSCLC)
Functional studies in NSCLC cells have demonstrated that depletion of ADCK2 through shRNA or CRISPR/Cas9 knockout significantly reduces cell viability, proliferation, cell cycle progression, and cell mobility, while inducing apoptosis . At the molecular level, ADCK2 depletion disrupts mitochondrial functions in NSCLC cells, resulting in cytochrome C release, mitochondrial depolarization, DNA damage, and ATP reduction . These effects are associated with inactivation of the Akt-mTOR signaling pathway, a key regulator of cell growth and survival . In vivo studies have confirmed that ADCK2 silencing or knockout significantly hinders NSCLC xenograft growth in nude mice .
ADCK2 in Melanoma
Interestingly, ADCK2 appears to play a contrasting role in melanoma compared to NSCLC. Analysis of data from the cBioPortal database indicates that higher intratumoral levels of ADCK2 correlate with better disease-specific survival in melanoma patients . This finding suggests a potential tumor-suppressive function of ADCK2 in melanoma, in stark contrast to its oncogenic role in NSCLC.
Functional studies in melanoma cells support this hypothesis. Knockdown of ADCK2 in SkMel28 melanoma cells leads to increased cell migration compared to control cells . Conversely, ADCK2 overexpression results in reduced migration capability . Similarly, ADCK2 knockdown enhances the invasion capacity of melanoma cells through a BME-coated matrix, while ADCK2 overexpression reduces invasion . These findings collectively suggest that ADCK2 inhibits the metastatic potential of melanoma cells, possibly by affecting MYL6 (Myosin Light Chain 6), a protein involved in cell motility .
Mechanism of Action
ADCK2 inhibitors comprise a class of chemical compounds specifically designed to suppress the activity of ADCK2 . These inhibitors function by binding to the active site or allosteric sites of the kinase, thereby preventing its phosphorylation activity . This inhibition interferes with the kinase's ability to transfer phosphate groups to substrate molecules, a critical step in the signaling cascades that regulate cellular metabolism and energy production . By blocking ADCK2's kinase function, these inhibitors effectively downregulate the pathways that rely on ADCK2, leading to a decrease in its downstream effects on mitochondrial processes .
The specificity of ADCK2 inhibitors is crucial for their therapeutic potential. These compounds must selectively target ADCK2 without affecting the vast array of other kinases within the cell . The design of ADCK2 inhibitors often involves the optimization of molecular interactions with the unique amino acid residues that line the binding pocket of ADCK2 . Some inhibitors achieve this by mimicking the ATP structure, as ADCK2 is an ATP-dependent kinase, while others are non-competitive and bind to sites distinct from the ATP binding site, inducing conformational changes that reduce kinase activity .
Current Developments in ADCK2-Targeted Therapies
The development of ADCK2 inhibitors is an emerging field in cancer therapeutics. Current research focuses on identifying compounds with high specificity and potency against ADCK2. The effectiveness of these inhibitors is typically assessed by measuring their impact on ADCK2's enzymatic activity in biochemical assays, where a decrease in substrate phosphorylation indicates effective inhibition .
Given the contrasting roles of ADCK2 in different cancer types, therapeutic strategies targeting this protein need to be cancer-specific. In NSCLC, where ADCK2 exhibits oncogenic properties, inhibiting ADCK2 could potentially reduce tumor growth and improve response to immunotherapy . In contrast, in melanoma, where ADCK2 appears to function as a tumor suppressor, strategies to enhance or restore ADCK2 expression or activity might be beneficial .
Potential Clinical Applications
In cancer therapy, the context-dependent roles of ADCK2 necessitate a personalized approach. For patients with NSCLC and high ADCK2 expression, ADCK2 inhibitors could potentially improve treatment outcomes, particularly when combined with immunotherapy . Conversely, for melanoma patients with low ADCK2 expression, strategies to enhance ADCK2 activity might help reduce metastatic potential and improve survival .
Expression Systems and Purification
Recombinant human ADCK2 is typically produced in bacterial expression systems, particularly E. coli . The expression construct usually includes the region spanning amino acids 128-389 of the human ADCK2 protein, with an N-terminal His Tag to facilitate purification . The recombinant protein has a molecular weight of approximately 28.7 kDa and can be purified to a high degree of homogeneity, with purity greater than 95% as determined by SDS-PAGE gel analysis .
The production process involves expression of the recombinant protein in E. coli, followed by cell lysis and purification using affinity chromatography . The purified protein is typically formulated as a lyophilized powder from a 0.2 μm filtered solution in PBS with 5% trehalose and 0.06% proclin, pH 7.4 . For use in research applications, the lyophilized protein can be reconstituted at 200 μg/mL in sterile distilled water .
Table 1: Characteristics of Recombinant Human ADCK2
| Parameter | Description |
|---|---|
| UniProt ID | Q7Z695 |
| Expression Region | 128-389 |
| Molecular Weight | 28.7 kDa |
| Tags | N-terminal His Tag |
| Purity | >95% by SDS-PAGE |
| Application | Western Blot, ELISA |
| Formulation | Lyophilized from PBS with 5% trehalose and 0.06% proclin, pH 7.4 |
| Reconstitution | 200 μg/mL in sterile distilled water |
| Storage | -20°C for 12 months (lyophilized); 2-8°C for 1 month after reconstitution |
| Alternative Names | AARF, aarF domain containing kinase 2, MGC20727, Putative ubiquinone biosynthesis protein AarF |
Applications in Research and Diagnostics
Recombinant ADCK2 has numerous applications in research and diagnostics. It can be used as a standard in Western blotting and ELISA for detecting and quantifying ADCK2 expression in biological samples . This application is particularly valuable for studies investigating ADCK2's role in cancer and other diseases, as well as for evaluating the efficacy of ADCK2-targeted therapies.
Recombinant ADCK2 can also serve as an antigen for generating specific antibodies against ADCK2. These antibodies can be used in various immunological techniques, including immunohistochemistry, immunofluorescence, and flow cytometry, to study ADCK2 expression and localization in cells and tissues. Additionally, recombinant ADCK2 can be used in protein-protein interaction studies to identify ADCK2's binding partners and understand its involvement in various signaling pathways.
Table 2: Contrasting Roles of ADCK2 in Different Cancer Types
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate your specific format requirements. Please indicate your preference in the order remarks, and we will prepare accordingly.
Note: All proteins are shipped with standard blue ice packs. If you require dry ice shipping, please notify us in advance, as additional charges will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
- ADCK4-related glomerulopathy represents a significant novel differential diagnosis for adolescents presenting with SRNS/FSGS and/or CKD of unknown origin PMID: 25967120
Q&A
What is ADCK2 and what are its fundamental characteristics?
ADCK2, or aarF Domain Containing Kinase 2, is a mitochondrial protein with an approximate mass of 74 kDa that belongs to the protein kinase superfamily within the ADCK protein kinase family . It plays critical roles in fatty acid metabolism and coenzyme Q biosynthesis . While classified as a potential protein kinase, it remains partially uncharacterized, with uncertainty regarding its specific kinase activity and substrate preferences (whether it phosphorylates Ser, Thr, or Tyr residues) .
Structurally, the recombinant form typically studied encompasses amino acids 128-389 of the human protein and is commonly expressed in Escherichia coli for research purposes . ADCK2 represents an important focus for the Illuminating the Druggable Genome (IDG) program due to its status as an understudied protein with significant physiological relevance .
How is ADCK2 involved in mitochondrial function?
ADCK2 plays a crucial role in mitochondrial function through multiple mechanisms:
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It facilitates the mitochondrial import of Coenzyme Q (CoQ) precursors, which are essential for the electron transport chain function .
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It supports fatty acid beta-oxidation, a key metabolic pathway that breaks down fatty acids to generate acetyl-CoA for the TCA cycle .
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Its depletion in cells disrupts normal mitochondrial functions, leading to several observable effects including:
This is further evidenced by studies showing that ADCK2 haploinsufficiency in humans is associated with mitochondrial myopathy characterized by lipid droplet accumulation in skeletal muscle, indicating impaired lipid metabolism .
What cellular pathways and metabolic processes involve ADCK2?
ADCK2 participates in several interconnected cellular pathways:
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Fatty acid metabolism: ADCK2 is integral to fatty acid beta-oxidation, with its deficiency leading to impaired lipid oxidation .
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Coenzyme Q biosynthesis: It facilitates the import of CoQ precursors into mitochondria, supporting electron transport chain function .
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Mitochondrial energy production: ADCK2 depletion results in ATP reduction, indicating its importance in cellular energy homeostasis .
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Cell survival and proliferation pathways: In NSCLC cells, ADCK2 depletion inactivates the Akt-mTOR signaling pathway, which normally promotes cell growth and survival .
These pathways highlight ADCK2's position at the intersection of energy metabolism, mitochondrial function, and cellular growth regulation.
What disease associations have been identified for ADCK2?
ADCK2 has been implicated in several disease contexts:
These disease associations highlight ADCK2's significance as both a potential biomarker and therapeutic target, particularly in cancer, where its depletion significantly hinders NSCLC xenograft growth in mouse models .
What is the expression pattern of ADCK2 in normal and disease tissues?
ADCK2 displays differential expression patterns between normal and disease states:
In normal tissues:
In disease tissues:
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ADCK2 is significantly overexpressed in NSCLC compared to normal lung epithelial tissues, as demonstrated by TCGA (The Cancer Genome Atlas) data for both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)
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This overexpression has been confirmed in:
Notably, bioinformatics analyses have revealed that high ADCK2 expression correlates with changes in the tumor immune microenvironment, including reduced infiltration of CD8+ T cells, eosinophils, and mast cells .
What experimental models and methodologies are recommended for studying ADCK2 function?
Researchers investigating ADCK2 have employed several experimental models and techniques:
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Cell Culture Models:
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Gene Manipulation Techniques:
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In Vivo Models:
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Functional Assays:
These methodologies provide a comprehensive toolkit for investigating ADCK2's molecular functions and physiological roles.
How can ADCK2 expression be effectively manipulated in experimental settings?
Based on successful approaches documented in the literature, researchers can manipulate ADCK2 expression through:
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ADCK2 Knockdown via shRNA:
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ADCK2 Knockout via CRISPR/Cas9:
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ADCK2 Overexpression:
These approaches have been successfully employed in various cell types, including NSCLC cell lines and primary cells, making them broadly applicable across research contexts.
What is known about ADCK2's role in cancer, particularly NSCLC?
ADCK2 appears to play a significant oncogenic role in NSCLC, supported by multiple lines of evidence:
These findings collectively identify ADCK2 as a potential therapeutic oncotarget in NSCLC, with its depletion showing anti-cancer effects through multiple mechanisms.
How does ADCK2 interact with mitochondrial metabolism and the coenzyme Q biosynthesis pathway?
ADCK2's interactions with mitochondrial metabolism and coenzyme Q biosynthesis occur through several mechanisms:
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Mitochondrial CoQ Precursor Import:
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Fatty Acid β-Oxidation:
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Energy Production:
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Mitochondrial Membrane Integrity:
While the exact molecular mechanisms remain to be fully elucidated, these observations underscore ADCK2's integral role in mitochondrial metabolism and function, particularly in the context of lipid metabolism and energy production.
What techniques are most effective for studying ADCK2 protein interactions and enzymatic activity?
Based on current research approaches, several techniques are particularly valuable for investigating ADCK2:
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Protein-Protein Interaction Analysis:
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Co-immunoprecipitation (Co-IP) to identify binding partners
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Proximity labeling techniques (BioID, APEX) for detecting transient or weak interactions in the mitochondrial environment
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Yeast two-hybrid screening for potential interactors
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Enzymatic Activity Assessment:
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In vitro kinase assays using recombinant ADCK2 protein to test potential substrates
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Phosphoproteomics analysis comparing wild-type to ADCK2-depleted cells
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ATP consumption assays to measure potential kinase activity
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Structural Studies:
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Functional Assessment:
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Metabolic flux analysis to measure changes in fatty acid oxidation
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CoQ level quantification using HPLC in ADCK2-manipulated cells
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Mitochondrial function assays (oxygen consumption rate, extracellular acidification rate)
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The recombinant human ADCK2 protein available from commercial sources (in the 128 to 389 amino acid range) provides a valuable tool for many of these investigations, particularly for in vitro studies of protein function and interactions .
What are the challenges in researching ADCK2 and how might they be addressed?
Researchers investigating ADCK2 face several challenges that require specific methodological approaches:
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Uncharacterized Enzymatic Activity:
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Challenge: The precise enzymatic function of ADCK2 remains unclear, including whether it has protein kinase activity and what substrates it might phosphorylate
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Solution: Employ comprehensive phosphoproteomic screening comparing wild-type and ADCK2-deficient cells; use chemical proteomics approaches to identify binding partners
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Mitochondrial Localization:
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Challenge: Mitochondrial proteins are often difficult to study due to the organelle's unique environment
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Solution: Use mitochondria-specific isolation techniques and targeted assays; employ proximity labeling methods specifically designed for mitochondrial environments
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Functional Redundancy:
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Challenge: ADCK family members may have overlapping functions
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Solution: Design studies that consider the entire ADCK family; use simultaneous knockdown/knockout approaches to identify compensatory mechanisms
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Disease Relevance Assessment:
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Understudied Protein Status:
Addressing these challenges requires interdisciplinary approaches and careful experimental design, ideally leveraging the resources developed through programs specifically targeting understudied proteins like ADCK2.
How does ADCK2 haploinsufficiency impact cellular and organismal physiology?
ADCK2 haploinsufficiency has significant pathophysiological consequences:
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Mitochondrial Myopathy Development:
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Liver Dysfunction:
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Impaired Fatty Acid Metabolism:
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Mitochondrial Function:
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Energy Production Deficits:
Studies using heterozygous Adck2 mouse models have provided valuable insights into these mechanisms, offering a platform for further investigation of potential therapeutic interventions for ADCK2-related disorders .
What is the potential of ADCK2 as a therapeutic target in cancer and other diseases?
ADCK2 shows considerable promise as a therapeutic target, particularly in cancer:
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Cancer Therapy Potential:
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Mechanisms Supporting Target Validity:
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Biomarker Potential:
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Mitochondrial Disease Applications:
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Target Development Status:
These findings collectively position ADCK2 as a molecular oncotarget with particular relevance to NSCLC, while also suggesting broader applications in mitochondrial disorders.
What is the current state of ADCK2 research and future directions?
ADCK2 research currently stands at an interesting intersection of basic science discovery and translational potential. As an understudied member of the aarF domain-containing protein kinase family, ADCK2 has established roles in mitochondrial function, fatty acid metabolism, and coenzyme Q biosynthesis, yet many aspects of its molecular function remain to be characterized .
The strongest evidence for ADCK2's functional significance comes from two seemingly distinct research areas:
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Cancer biology, where ADCK2 overexpression promotes NSCLC growth and correlates with poor clinical outcomes and immunotherapy resistance
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Mitochondrial disease, where ADCK2 haploinsufficiency leads to mitochondrial myopathy, liver dysfunction, and impaired lipid metabolism
Future research directions should focus on:
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Determining ADCK2's precise enzymatic activity and substrate specificity
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Identifying its protein interaction network within mitochondria
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Developing potential therapeutic approaches targeting ADCK2 in cancer
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Investigating compensatory mechanisms that might be activated in ADCK2-deficient states
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Exploring connections between ADCK2 and other mitochondrial disorders
