ANKRD54 Human

What is the basic structure and function of human ANKRD54?

ANKRD54, also known as LIAR, is a 34.9 kDa protein (323 amino acids) containing 4 ankyrin repeat domains. It plays a crucial role in regulating intracellular signaling events associated with erythroid terminal differentiation. The protein functions primarily through protein complex binding and protein kinase regulator activity. ANKRD54 is encoded by a gene located on chromosome 22q13.1 (Gene ID: 129138), with mRNA RefSeq NM_138797 and protein RefSeq NP_620152 .

Which key protein interactions characterize ANKRD54 function?

ANKRD54 interacts with several proteins in a specific manner to perform its cellular functions:

• It binds to LYN via its ankyrin repeat region, specifically interacting with LYN's SH3-domain in an activation-independent manner

• It forms a multiprotein complex with LYN and HCLS1

• It also interacts with TSN2, VAV1, DBNL, and LASP1

These interactions suggest ANKRD54 serves as a scaffolding protein in signaling complexes, particularly those involved in hematopoietic cell development.

How does subcellular localization affect ANKRD54 function?

ANKRD54 contains both nuclear localization signals (NLS) and nuclear export signals (NES), enabling it to shuttle between the nucleus and cytoplasm. This localization is dynamically regulated by phosphorylation events. The protein's function likely depends on its subcellular compartmentalization, with different roles in nuclear and cytoplasmic contexts. Studies using eGFP-tagged Ankrd54 have confirmed the functionality of these localization signals .

What mechanisms regulate ANKRD54 nuclear-cytoplasmic shuttling?

The subcellular localization of ANKRD54 is regulated primarily through phosphorylation by protein kinase C delta (PKCδ). Research has demonstrated that:

• Activation of PKC kinases using phorbol 12-myristate 13-acetate (PMA) promotes nuclear export of ANKRD54 and correlates with increased phosphorylation

• Co-expression of active PKCδ specifically promotes both phosphorylation and cytoplasmic localization of ANKRD54

• Alanine mutation of specific serine residues in the amino-terminal region (Ser14, Ser17, Ser18, Ser19) reduces both PMA-induced cytoplasmic localization and phosphorylation

Multiple reaction monitoring (MRM) proteomic analysis has specifically identified phosphorylation of the Ser18 residue in response to PMA stimulation, suggesting this site plays a critical role in localization control .

How can researchers effectively study ANKRD54 phosphorylation states?

Researchers can employ several methodologies to study ANKRD54 phosphorylation:

• Phos-tag™ gel retardation assays - This technique allows for separation of phosphorylated and non-phosphorylated forms of the protein, enabling quantitative assessment of phosphorylation levels

• Multiple-reaction-monitoring (MRM) proteomic analysis - This targeted mass spectrometry approach can identify specific phosphorylated residues, as demonstrated for Ser18 in ANKRD54

• Site-directed mutagenesis - Creating serine-to-alanine mutations (S14A/S17A/S18A/S19A) can help determine which phosphorylation sites are functionally important

• Pharmacological approaches - Using kinase activators (PMA), inhibitors (staurosporin), or phosphatase inhibitors (calyculin A) to manipulate phosphorylation states

These complementary approaches provide robust data on ANKRD54 phosphorylation dynamics and their functional consequences.

What is the relationship between ANKRD54 and the Lyn tyrosine kinase?

ANKRD54 and Lyn tyrosine kinase have a complex functional relationship that can be studied using various experimental approaches:

• Co-immunoprecipitation assays show that PMA stimulation enhances the interaction between ANKRD54 and Lyn

• Fluorescence microscopy using eGFP-tagged ANKRD54 and antibody detection of Lyn demonstrates enhanced co-localization in the cytoplasm upon PMA stimulation

• The interaction occurs via the ankyrin repeat region of ANKRD54 and the SH3-domain of Lyn, independent of Lyn's activation status

This interaction suggests ANKRD54 may regulate Lyn's signaling activities or subcellular localization, particularly during erythroid differentiation.

What expression systems are optimal for recombinant ANKRD54 production?

For recombinant ANKRD54 production, E. coli expression systems have been successfully employed. The following specifications have proven effective:

This approach yields functional ANKRD54 protein suitable for biochemical and structural studies .

How can researchers effectively visualize ANKRD54 subcellular localization?

To study ANKRD54 subcellular localization, researchers should consider these methodological approaches:

• Fluorescent protein tagging - N-terminal eGFP fusion has been validated to maintain functional NLS and NES signals

• Confocal microscopy or deconvolution systems - For high-resolution imaging of subcellular compartments

• Quantitative analysis - Measure nuclear:cytoplasmic ratios by analyzing 10 nuclear and 10 cytoplasmic sub-compartments per cell across 50-100 cells

• Co-localization studies - Use TRITC-labeled phalloidin to visualize F-actin as a cytoplasmic marker and Hoechst for nuclear staining

• Statistical analysis - Apply student's t-test or ANOVA with two-tailed analysis and orthogonal comparisons across three biological replicates

This comprehensive imaging approach provides robust data on ANKRD54 localization dynamics.

What analytical techniques best characterize ANKRD54 phosphorylation sites?

For precise characterization of ANKRD54 phosphorylation sites, researchers should employ:

• Chymotrypsin digestion - Optimal for generating peptides containing the key phosphorylation sites

• Electrospray ionization mass spectrometry - Using an Ultimate 3000 nano HPLC system coupled to a 4000 Q TRAP mass spectrometer

• Peptide resolution - Using a C18 PepMap100 column with a linear gradient of water/acetonitrile with 0.1% formic acid

• Multiple reaction monitoring (MRM) - Generate transitions for putative phosphopeptides using Skyline software

• Control peptides - Include non-phosphorylated peptides (e.g., sequence QQDVEPRDEL, corresponding to amino acids 72-81) as internal controls

This analytical pipeline successfully identified Ser18 as a key phosphorylation site in ANKRD54 and can be applied to discover additional modification sites.

How does ANKRD54 contribute to erythroid terminal differentiation?

ANKRD54 regulates intracellular signaling during erythroid terminal differentiation through:

• Modulation of kinase activity - Acting as a protein kinase regulator, potentially affecting differentiation signals

• Protein scaffolding - Forming complexes with key signaling molecules like LYN and HCLS1

• Dynamic subcellular localization - Shuttling between nucleus and cytoplasm based on phosphorylation state, potentially transporting binding partners

• Signal integration - Connecting various pathways through interactions with multiple partners (TSN2, VAV1, DBNL, LASP1)

To study these functions, researchers should design experiments that track ANKRD54 localization and interaction partners during different stages of erythroid differentiation.

What experimental models best capture ANKRD54 physiological functions?

When designing experiments to study ANKRD54 physiological roles, researchers should consider:

• Cell models - HEK293T cells provide a reliable system for protein expression and localization studies, while hematopoietic cell lines would better represent the native context

• Expression constructs - Full-length murine or human ANKRD54 cDNA constructs, with careful attention to NLS and NES elements

• Protein tagging - N-terminal eGFP fusion has been validated, with mCherry-tagged interaction partners for co-localization studies

• Functional mutants - Create site-directed mutants targeting:

  • Phosphorylation sites (S14A/S17A/S18A/S19A)

  • NLS elements (KKRL111AALA, RR98AA, D98-114)

  • NES sequences (LSL289ASA)

These experimental approaches provide complementary insights into ANKRD54 function in relevant biological contexts.

How might ANKRD54 be implicated in hematological disorders?

Given ANKRD54's role in erythroid differentiation and signaling, future research should investigate:

• Expression profiling - Examine ANKRD54 expression levels in various hematological disorders, particularly those affecting erythroid lineages

• Mutation screening - Analyze patient samples for mutations in ANKRD54, particularly in the ankyrin repeat domains or phosphorylation sites

• Functional consequences - Determine how alterations in ANKRD54 affect LYN signaling and erythroid differentiation

• Therapeutic potential - Explore whether modulating ANKRD54 function or its interactions could provide treatment avenues

This translational approach could connect fundamental ANKRD54 biology to clinical applications.

What technologies will advance our understanding of ANKRD54 structure-function relationships?

Emerging technologies likely to enhance ANKRD54 research include:

• Cryo-electron microscopy - To determine the structure of ANKRD54 within its multiprotein complexes

• Proximity labeling techniques (BioID, APEX) - To comprehensively map the ANKRD54 interactome in different cellular contexts

• Single-cell phosphoproteomics - To capture the dynamics of ANKRD54 phosphorylation in heterogeneous cell populations

• CRISPR-Cas9 genome editing - To create precise modifications in endogenous ANKRD54, avoiding potential artifacts of overexpression

• Optogenetic approaches - To control ANKRD54 localization or interactions with temporal precision

These advanced approaches will provide deeper insights into how ANKRD54 structure relates to its diverse cellular functions.