DCAF7 Human

What is the structural characterization of DCAF7?

DCAF7 is characterized as an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity of its own . WD40 domains typically contain several WD40 repeats, which are sequences of 44–60 amino acids in length that often harbor a defining tryptophan-aspartate (WD) dipeptide . These repeats form a circularized β-propeller structure that provides multiple binding surfaces for diverse protein–protein interactions, which is consistent with DCAF7's role as a scaffolding protein . This structural arrangement is common among numerous scaffold proteins and facilitates DCAF7's function in mediating protein interactions rather than acting as an enzyme itself.

What are the main protein interactions of DCAF7?

DCAF7 has been identified as a binding partner for several key proteins:

• Kinase interactions: DCAF7 binds to DYRK1A, DYRK1B, and HIPK2 but notably not to HIPK1 or class 2 DYRKs (DYRK2, DYRK3, DYRK4) .

• Insulin signaling components: DCAF7 interacts with IRS1 (Insulin Receptor Substrate 1) and the p85 subunit of PI3K, functioning as a scaffold in insulin signaling .

• Viral proteins: DCAF7 mediates the interaction between adenovirus E1A protein and DYRK1A/HIPK2 .

These interactions are highly specific and evolutionarily conserved. The binding interfaces between DCAF7 and its partners have been mapped to specific regions in these proteins, demonstrating the specialized nature of these interactions .

How is DCAF7 expression or function conserved across species?

DCAF7's function appears to be highly conserved throughout evolution. Research has demonstrated that:

• Human DCAF7 can interact with Xenopus DYRK1B .

• Mouse DYRK1A, human DYRK1B, and Xenopus DYRK1B can all bind to zebrafish DCAF7 .

• The DCAF7 binding motif identified in DYRK1A is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio, and even the slime mold Dictyostelium discoideum .

This high degree of evolutionary conservation suggests that DCAF7 plays a fundamental role in cellular processes that have been maintained throughout evolution, emphasizing its biological significance . Studies in various model systems including Drosophila (where DCAF7 is known as Wap) show that loss or mutation of DCAF7 is associated with growth retardation, confirming its essential role across diverse species .

How does DCAF7 function in the insulin signaling pathway?

DCAF7 acts as a scaffold for IRS1-PI3K that is required for insulin signaling to AKT and FOXO1 inhibition . Experimental evidence supports this role:

• Scaffold function: DCAF7 interacts with IRS1 and the p85 subunit of PI3K, suggesting it brings these proteins together to facilitate signaling .

• Effect on AKT phosphorylation: Knockdown of DCAF7 significantly diminishes insulin-stimulated AKT S473 and T308 phosphorylation, while not affecting ERK phosphorylation, demonstrating specificity for the PI3K-AKT pathway .

• IRS1 phosphorylation: Following DCAF7 knockdown, phosphorylation of S612 in IRS1 increases. Since S612 is a known inhibitory site, this may explain the observed decrease in insulin signaling .

The role of DCAF7 as an IRS1 scaffolding protein is further supported by the finding that knockdown of DCAF7 decreases the interaction of IRS1 with DYRK1A, demonstrating its importance in facilitating protein complexes necessary for proper signaling .

What is DCAF7's relationship with DYRK1A and how does it affect substrate phosphorylation?

DCAF7 serves as an adaptor protein for DYRK1A and mediates interactions between DYRK1A and its substrates:

• Complex formation: DCAF7 binds to a conserved 12-amino acid motif in the N-terminal domain of DYRK1A, forming a stable complex at physiological expression levels .

• Substrate recruitment: DCAF7 functions as a substrate recruiting subunit for DYRK1A . This is evidenced by experiments showing that DCAF7 is required for the hyperphosphorylation of the adenovirus E1A protein in DYRK1A overexpressing cells .

• Binding interface: The interaction does not depend on DYRK1A's catalytic activity, as kinase-deficient mutants maintain DCAF7 binding .

This relationship appears to be essential for proper DYRK1A function, as DCAF7 helps bring together DYRK1A and its substrates, facilitating efficient phosphorylation .

How does DCAF7 affect cell cycle regulation and proliferation?

DCAF7 plays a critical role in regulating cell proliferation through its effects on cell cycle progression:

• Cell number effects: Knockdown of DCAF7 significantly reduces cell number over time, with measurable differences observed by days 4-5 post-transfection .

• Cell cycle arrest: Flow cytometry analysis reveals that DCAF7 knockdown induces cell cycle arrest specifically at the G2 phase, similar to the effect of Dinaciclib, a cyclin-dependent kinase inhibitor .

• Molecular mechanism: DCAF7 knockdown leads to:

  • Reduced AKT phosphorylation

  • Enhanced nuclear localization of FOXO1

  • Significant increase in expression of FOXO1-target genes involved in G2 cell cycle arrest (GADD45A and CCNG2)

This regulatory mechanism appears to be conserved, as studies in Drosophila show that knockdown of the DCAF7 ortholog (wap) reduces wing area and cell number through a dFOXO-dependent mechanism, mirroring observations in flies lacking chico (the IRS ortholog) .

What methods can be used to study DCAF7-mediated protein-protein interactions?

Several experimental approaches have proven effective for investigating DCAF7's interactions:

• Co-immunoprecipitation (co-IP): This has been successfully used to demonstrate interactions between DCAF7 and its partners. Both overexpressed tagged proteins and endogenous proteins can be immunoprecipitated to verify interactions . For example, immunoprecipitation of endogenous DYRK1A from HeLa cells confirmed interaction with DCAF7 at physiological expression levels .

• GST pulldown assays: Recombinant GST-fused proteins (like E1A-X2) can be used as bait to pull down DCAF7 and its binding partners from cell lysates . This approach helps identify direct versus indirect interactions.

• In vitro translation: Producing proteins like GFP-DCAF7 by in vitro translation for use as prey in GST pulldown experiments can further verify direct binding to partners like E1A and DYRK1A .

• Deletion mapping: Creating truncated constructs of DCAF7's binding partners has been effective in mapping specific binding domains. For example, this approach identified that amino acids 1-135 of HIPK2 contain the DCAF7 binding site, while a shorter construct (amino acids 1-114) does not bind DCAF7 .

• Site-directed mutagenesis: Point mutations within potential binding interfaces can confirm critical residues. The substitution of Thr125 by proline abolished DCAF7 binding to HIPK2, demonstrating the importance of this residue .

These methodologies, especially when used in combination, provide robust evidence for DCAF7's role as an adaptor protein in various complexes.

What approaches can be used to study DCAF7's role in cell proliferation?

Researchers have employed several techniques to investigate DCAF7's impact on cell proliferation:

• siRNA knockdown: Efficient reduction of DCAF7 protein abundance can be achieved using siRNA transfection, with effects observable up to 5 days post-transfection .

• Cell counting assays: Direct measurement of cell number over time following DCAF7 knockdown demonstrates its effect on proliferation .

• Flow cytometry:

  • S10-phosphorylated histone H3 (a marker of mitosis) combined with propidium iodide (PI) to assess DNA content can determine cell cycle phase distribution

  • Annexin V and PI staining can be used to assess cell death rates following DCAF7 manipulation

• Western blotting: Analysis of phosphorylation status of key signaling proteins (AKT, ERK, IRS1) after insulin stimulation in DCAF7-depleted cells reveals pathway-specific effects .

• Immunofluorescence: Subcellular localization of factors like FOXO1 can be visualized following DCAF7 knockdown to determine nuclear versus cytoplasmic distribution .

• RT-qPCR: Expression analysis of FOXO1-target genes involved in cell cycle regulation (GADD45A, CCNG2, CDKN1B, RBL2) provides mechanistic insight into how DCAF7 affects proliferation .

These methodological approaches provide complementary data to establish DCAF7's role in regulating cell proliferation through specific signaling pathways.

How does DCAF7 function in relation to the DDB1-Cul4 E3 ubiquitin ligase complex?

Although DCAF7's name suggests a function in E3 ubiquitin ligase complexes, the research presents a nuanced picture:

• Potential role: DCAF7 is described as a substrate-specific adaptor of the DDB1-Cul4 E3 ubiquitin ligase complex, potentially promoting proteasomal degradation of targeted proteins .

• Proteasome-independent functions: DCAF7 also regulates interactor levels independent of the proteasome . For instance, knockdown of DCAF7 did not affect IRS1 protein abundance, suggesting a functional role for the DCAF7-IRS1 interaction rather than mediating protein stability .

• Research limitations: While DCAF7's name (DDB1 and CUL4 associated factor 7) refers to its identification in DDB1 complexes and its deduced function as a substrate receptor in CLR4 E3 ubiquitin ligase complexes, this specific role has not been fully experimentally verified .

Understanding this aspect of DCAF7 function remains an area where additional research is needed to clarify its precise role in protein degradation pathways versus its adaptor/scaffold functions.

What are the distinguishing features of DCAF7's binding to different kinases?

DCAF7 shows remarkable specificity in its kinase interactions:

• Class 1 vs. Class 2 DYRKs: DCAF7 binds to class 1 DYRKs (DYRK1A, DYRK1B) but not to class 2 DYRKs (DYRK2, DYRK3, DYRK4) . This specificity correlates with sequence conservation in the N-terminal region of class 1 DYRKs, which diverges in class 2 DYRKs.

• HIPK1 vs. HIPK2: Despite HIPK1 and HIPK2 being closely related with the same domain architecture, DCAF7 specifically binds to HIPK2 but not HIPK1 . This selectivity was traced to critical residues in the binding interface - specifically, the presence of a proline in HIPK1 at a position corresponding to Thr125 in HIPK2 disrupts binding .

• Binding motif conservation: The DCAF7 binding motif consists of 12 amino acids in DYRK1A (amino acids 93-104) with a similar sequence essential for DCAF7 binding to HIPK2 .

• Kinase activity independence: The interaction of DCAF7 with both DYRK1A and HIPK2 does not depend on the catalytic activity of these kinases, as binding is maintained with kinase-deficient mutants .

This binding specificity highlights DCAF7's evolved role as a selective adaptor for specific kinases, suggesting specialized functions in distinct signaling pathways.

How to resolve contradictory findings regarding DCAF7's role in cell death versus cell cycle arrest?

The literature presents some nuanced findings regarding DCAF7's effects on cell viability versus proliferation:

• Temporal progression: DCAF7 knockdown initially causes cell cycle arrest (observable early) followed by compromised cell viability at later timepoints (days 4-5 post-transfection) . This suggests a sequential rather than contradictory relationship between these observations.

• Methodological considerations:

  • Cell cycle analysis using phospho-histone H3 and PI staining by flow cytometry can distinguish between cell cycle effects and cell death

  • Complementary assays for cell death (LDH release, Annexin V/PI staining) help determine the extent of apoptosis/necrosis versus cell cycle arrest

• Experimental design recommendations:

  • Conduct time-course experiments to distinguish early versus late effects

  • Include appropriate positive controls (e.g., Dinaciclib for cell cycle arrest at G2, Staurosporine for cell death)

  • Use multiple cell lines to ensure findings are not cell-type specific

  • Employ rescue experiments with wild-type DCAF7 to confirm specificity of knockdown effects

• Mechanistic relationship: The data suggests that prolonged G2 arrest following DCAF7 knockdown eventually leads to cell death, consistent with known cellular responses to sustained cell cycle checkpoint activation .

Understanding this relationship requires careful experimental design with appropriate time points and complementary methodologies to distinguish primary from secondary effects.

What are the key unanswered questions about DCAF7 function?

Despite significant progress in understanding DCAF7, several important questions remain:

• E3 ligase function: Though DCAF7 is named as a CUL4 associated factor, its precise role in ubiquitin ligase complexes requires further verification and characterization .

• Substrate specificity: While DCAF7 functions as a substrate recruiting subunit for DYRK1A and HIPK2, the complete set of substrates it helps recruit remains to be fully characterized .

• Tissue-specific functions: Most studies have used cell lines or model organisms; the tissue-specific roles of DCAF7 in human physiology and disease remain largely unexplored.

• Disease relevance: The potential contribution of DCAF7 dysfunction to human diseases, particularly those involving insulin signaling, cell cycle regulation, or DYRK1A function (e.g., neurodevelopmental disorders) warrants investigation.

• Therapeutic targeting: Given DCAF7's role as an adaptor protein in multiple signaling pathways, its potential as a therapeutic target for modulating these pathways remains an open question.

Addressing these questions will require integrative approaches combining structural biology, proteomics, genetic models, and clinical studies.

What methodological approaches might advance our understanding of DCAF7?

Future research on DCAF7 could benefit from several cutting-edge approaches:

• CRISPR-based technologies:

  • CRISPR-Cas9 knockout and knockin models to study DCAF7 function in various cell types

  • CRISPRi/CRISPRa for tunable control of DCAF7 expression

  • Base editors for introducing specific mutations in binding interfaces

• Proximity labeling proteomics:

  • BioID or APEX2 fusion proteins to identify the complete DCAF7 interactome under various cellular conditions

  • TurboID for temporal resolution of dynamic interactions

• Structural biology:

  • Cryo-EM structures of DCAF7 complexes with various binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Single-cell approaches:

  • Single-cell RNA-seq to understand cell-type-specific responses to DCAF7 modulation

  • Live-cell imaging with fluorescent reporters to visualize DCAF7-dependent signaling dynamics

• Animal models:

  • Conditional tissue-specific knockout models to assess DCAF7 function in specific physiological contexts

  • Humanized mouse models expressing human DCAF7 variants

These methodological advances would provide deeper insights into DCAF7's structure, interactions, and functions in normal physiology and disease states.