ACK1 Antibody

What is ACK1 and what are its primary biological functions?

ACK1, also designated as TNK2 (tyrosine kinase, non-receptor, 2), belongs to the protein kinase superfamily and functions as both a non-receptor tyrosine-protein and serine/threonine-protein kinase. This versatile signaling molecule plays critical roles in multiple cellular processes including cell spreading and migration, cell survival, and proliferation . As a downstream effector of CDC42, ACK1 mediates CDC42-dependent cell migration by transducing extracellular signals to cytosolic and nuclear effectors .

The protein contains multiple functional domains including a tyrosine kinase domain, sterile α motif domain, Src homology domain 3 (SH3), Cdc42- and Rac-interactive binding (CRIB) domain, a clathrin-interacting region, a WW domain, a MIG6 homology region that binds EGFR, and a ubiquitin association (UBA) domain . This complex domain structure enables ACK1 to function as a central signaling integrator for receptor tyrosine kinases, particularly in EGFR trafficking and degradation pathways .

What applications can ACK1 antibody be used for in research settings?

Based on validated experimental data, ACK1 antibody (14304-1-AP) has been successfully employed in multiple research applications:

The antibody has demonstrated positive Western blot detection in multiple cell lines (SH-SY5Y, HepG2, HT-1080, A431, HeLa, A549) and mouse brain tissue, providing versatility across different experimental systems . For immunohistochemistry applications, the antibody has been successfully used with human breast cancer tissue, with specific antigen retrieval recommendations available to maximize detection sensitivity .

What are the optimal experimental conditions for using ACK1 antibody?

For optimal experimental results, researchers should consider the following application-specific dilutions and conditions:

It is strongly recommended that researchers titrate the antibody in each specific testing system to obtain optimal results, as optimal conditions may be sample-dependent . The antibody is provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and should be stored at -20°C where it remains stable for one year after shipment .

What is the molecular weight of ACK1 protein and how does this affect antibody detection?

An interesting discrepancy exists between the calculated and observed molecular weights of ACK1 protein. While the calculated molecular weight is 115 kDa, the observed molecular weight in experimental settings is typically 70 kDa . This difference is important for researchers to understand when interpreting Western blot results, as the detection band may appear at a lower molecular weight than theoretical predictions would suggest. This discrepancy could be attributed to post-translational modifications, alternative splicing, or proteolytic processing that occurs in vivo.

How does ACK1 contribute to autoimmune diseases like Systemic Lupus Erythematosus (SLE)?

Recent genetic evidence has identified an association between SLE and compound heterozygous deleterious variants in ACK1 (TNK2) and BRK non-receptor tyrosine kinases . Experimental studies demonstrate that inhibition of ACK1 or BRK increases circulating autoantibodies in mice and exacerbates glomerular IgG deposits in SLE mouse models .

The mechanism appears to involve impaired efferocytosis, specifically the MERTK-mediated anti-inflammatory response to apoptotic cells. In human induced pluripotent stem cell (hiPSC)-derived macrophages, ACK1 and BRK variants impair this process, potentially contributing to SLE pathogenesis .

Experimental data shows that in BALB/cByJ mice receiving ACK1 inhibitor AIM100:

• Mice developed a broad array of circulating anti-nuclear IgG antibodies including anti-histones, anti-chromatin, anti U1-snRNP, anti-SSA, and anti-Ku antibodies

• Inhibition of ACK1 worsened pristane-induced Lupus in wild-type mice

• Increased kidney glomerular deposits of IgG were observed in treated mice

These findings suggest that ACK1 deficiency may underlie or contribute to SLE development in genetically susceptible individuals depending on genetic and environmental context .

What are the key interacting partners of ACK1 in neuronal tissues?

Mass spectrometry approaches have identified novel ACK1-interacting proteins in brain tissue. Through immunoprecipitation assays coupled with LC-MS/MS analysis, researchers have confirmed a significant interaction between ACK1 and calcium/calmodulin-dependent protein kinase II alpha (CAMKII-α) .

Co-immunoprecipitation assays in adult brain samples followed by Western blot analysis revealed that:

• ACK1 antibodies co-precipitated CAMKII-α

• Reciprocally, CAMKII-α immunoprecipitates yielded ACK1

Co-immunolocalization experiments in hippocampal neuronal cultures demonstrated that CAMKII-α and ACK1 largely colocalize in most neuronal compartments, including soma, dendrites, and axons, though this co-localization was in some cases partial or incomplete . Interestingly, CAMKII-α was particularly enriched at the tips of developing dendrites and axons (growth cones), whereas ACK1 was not .

This interaction suggests potential roles for ACK1 in neuronal development, synaptic plasticity, or neuronal signaling that warrant further investigation.

What is the role of ACK1 in tumor formation and cancer progression?

Despite the frequent amplification and mutation of ACK1 (TNK2) in different human cancers, experimental evidence from knockout models presents a more complex picture regarding its role in tumorigenesis. In head-and-neck squamous cell carcinoma, 59% show low-level gain alterations of the TNK2 gene, and 13% show amplification, resulting in increased mRNA expression of ACK1 . In breast cancer, activated ACK1 correlates negatively with survival .

• ACK1 knockout mice showed no significant changes in tumor development in a skin cancer model

• Analysis of signaling pathways in tumors from control and ACK1 knockout mice revealed similar levels of pErk, pp130Cas, and E-cadherin, suggesting ACK1 is not critical for these pathways in this context

• CRISPR-generated ACK1-null breast cancer cell lines (including MDA-MB-231, 4T1, MCF7, T47D, and 67NR) showed no significant changes in morphology or in vitro growth

• EGF stimulation kinetics in T47D cells lacking ACK1 showed normal induction of phosphorylated forms of Erk and Akt

These findings suggest that despite its frequent alteration in human cancers, ACK1 may have a more context-dependent role in cancer progression than previously thought, or that compensatory mechanisms may exist in knockout models.

How should I optimize ACK1 antibody for co-immunoprecipitation experiments?

For successful co-immunoprecipitation experiments with ACK1 antibody, researchers should consider the following methodological approaches based on published protocols:

• Tissue/sample preparation: Brain homogenates or other tissue samples should be properly lysed in appropriate buffer conditions that preserve protein-protein interactions .

• Immunoprecipitation procedure:

  • Use proper antibody-to-protein ratio for efficient capture of ACK1 and its interacting partners

  • Include appropriate controls (IgG control, reverse immunoprecipitation)

  • For validation of interactions, perform reciprocal co-immunoprecipitation as demonstrated with ACK1 and CAMKII-α

• Detection methods:

  • Western blot analysis using specific antibodies against potential interacting partners

  • For comprehensive interactome analysis, consider tandem mass spectrometry approaches (LC-MS/MS) as successfully employed to identify ACK1-interacting proteins

• Validation of interactions:

  • Confirm interactions through alternative methods such as co-immunolocalization experiments in relevant cell types

  • As demonstrated with ACK1 and CAMKII-α, co-localization studies in neuronal cultures can provide spatial information about interaction contexts

How does ACK1 interact with EGFR signaling pathways?

ACK1 has been proposed to mediate EGF-induced EGFR degradation through multiple mechanisms:

• Binding to ubiquitin ligases via its UBA domain

• Association with clathrin-coated pits via its clathrin-interacting domain

• Regulation of EGFR trafficking to the p62/Next to BRCA1 gene 1 protein pre-autophagosome

• In T47D breast cancer cells, loss of ACK1 did not result in obvious changes in EGF-induced phosphorylation kinetics of Erk and Akt

• In MDA-MB-231 cells, EGF treatment did not result in significant changes in EGFR, pErk, Erk, pAkt, Akt, ppCas130, and pCas130 regardless of ACK1 status

These findings suggest that while ACK1 has the structural capacity to interact with EGFR pathways, its importance in mediating EGFR signaling may be cell-type dependent or compensated by other mechanisms in the cancer cell lines studied.

What controls should be included when using ACK1 antibody for immunoblotting?

When using ACK1 antibody for Western blot applications, researchers should include the following controls:

• Positive controls: Consider using lysates from cells known to express ACK1, such as SH-SY5Y, HepG2, HT-1080, A431, HeLa, or A549 cells, or mouse brain tissue, all of which have shown positive detection with the antibody .

• Negative controls:

  • Lysates from ACK1 knockout cells or tissues, if available

  • Samples treated with ACK1-targeting siRNA or shRNA

  • Pre-absorption of the antibody with the immunizing peptide

• Loading controls: Include appropriate loading controls such as β-actin, GAPDH, or total protein staining to ensure equal loading across samples.

• Molecular weight markers: Include appropriate molecular weight markers and note that ACK1 typically appears at 70 kDa despite a calculated molecular weight of 115 kDa .

• Antibody specificity validation: If possible, validate specificity through genetic models lacking ACK1 expression, as demonstrated in studies using ACK1 knockout mice and CRISPR-edited cell lines .

What experimental models are most appropriate for studying ACK1 function?

Based on the literature, several experimental models have proven valuable for studying ACK1 function:

• Cell culture models:

  • Neuronal cultures for studying ACK1's role in neuronal development and function

  • Breast cancer cell lines (MDA-MB-231, 4T1, MCF7, T47D, and 67NR) for investigating ACK1's role in cancer

  • Human induced pluripotent stem cell (hiPSC)-derived macrophages for studying efferocytosis and inflammatory responses

• Animal models:

  • ACK1 knockout mice for studying development and physiological functions

  • BALB/cByJ mice with ACK1 inhibitor treatment for autoimmunity studies

  • Pristane-induced lupus mouse model for studying ACK1's role in autoimmune disease

• Pharmacological approaches:

  • Chemical inhibitors of ACK1 such as AIM100 can be used to study acute inhibition effects versus genetic knockout compensation

Each model offers specific advantages depending on the research question, with genetic knockout models providing insights into developmental roles while pharmacological inhibition can reveal acute signaling functions that might be compensated in genetic models.

What are the most promising research directions for ACK1 antibody applications?

Based on current literature, several promising research directions emerge for ACK1 antibody applications:

• Autoimmune disease research: Further investigation into ACK1's role in efferocytosis and autoimmune disease pathogenesis, particularly in SLE and related conditions .

• Neuronal function: Exploring the functional significance of ACK1's interaction with CAMKII-α and other neuronal proteins, particularly in synaptic plasticity and neuronal development .

• Cancer biology context-dependency: Resolving the apparent contradiction between ACK1 amplification in human cancers and the limited phenotype of ACK1 knockout in experimental models .

• Signaling network analysis: Comprehensive mapping of ACK1's position in cellular signaling networks, particularly in relation to receptor tyrosine kinase pathways and CDC42-mediated processes .

• Therapeutic targeting: Evaluating the potential of ACK1 as a therapeutic target in specific disease contexts, with careful consideration of its context-dependent functions.