Phospho-CCDC88A (Ser1417) Antibody

What is CCDC88A and why is the Ser1417 phosphorylation site significant?

CCDC88A (Coiled-Coil Domain-Containing Protein 88A) encodes the actin-binding protein Girdin, which is expressed ubiquitously across human tissues with highest expression in brain and testis . The Girdin protein (216 kDa) has a complex domain architecture comprising:

• N-terminal microtubule-binding Hook domain (1-196 aa)

• Coiled-coil domain (196-1304 aa) involved in homodimerization

• C-terminal region containing multiple functional domains:

  • Gα-binding domain (GBD) (1343-1424 aa)

  • PI4P-binding site (1390-1408 aa)

  • AKT1 phosphorylation site at Ser1417

  • Actin-binding domain (1623-1870 aa)

The Ser1417 site is particularly important because its phosphorylation by AKT1 triggers Girdin's relocalization to lamellipodia where it crosslinks actin filaments . This phosphorylation is critical for cell migration and cytoskeletal reorganization, making it a key regulatory site for Girdin's biological functions .

Thorough validation requires multiple approaches:

• Phosphopeptide ELISA: Compare binding to phosphorylated vs. non-phosphorylated peptides of the same sequence to confirm phospho-specificity .

• Western blot analysis: Test using cell lysates from:

  • Untreated control cells

  • Growth factor-stimulated cells (e.g., EGF stimulation promotes phosphorylation)

  • Cells treated with PI3K/AKT inhibitors (should reduce phosphorylation)

  • CCDC88A-knockdown cells using siRNA (should show reduced signal)

• Immunoprecipitation coupled with western blotting: Precipitate with anti-CCDC88A antibody followed by detection with phospho-specific antibody .

• Validation by molecular weight: The detected band should be at approximately 216 kDa (calculated molecular weight) .

What methodologies are recommended for studying CCDC88A's role in cancer cell migration and invasion?

CCDC88A has been implicated in cancer cell migration and invasion, particularly in pancreatic ductal adenocarcinoma (PDAC). The following methods have yielded significant insights:

• siRNA knockdown: Transfection with 80 pmol siRNA mixture targeting CCDC88A for 48 hours effectively suppresses expression .

• Rescue experiments: Transfection of CCDC88A-rescue construct into siRNA-transfected cells to verify specificity of observed phenotypes .

• Migration and invasion assays:

  • Transwell migration assay

  • Two-chamber Matrigel invasion assay

  Key finding: CCDC88A knockdown significantly inhibited cell migration and invasion in S2-013 and PANC-1 pancreatic cancer cell lines without affecting cell growth in MTT assays .

• Confocal microscopy analysis:

  • Analyze peripheral actin structures in membrane ruffles

  • Study fibronectin-mediated formation of membrane protrusions

  • Track CCDC88A localization in cell protrusions

• Co-localization studies: Analyze CCDC88A co-localization with actin filaments in cell protrusions using immunofluorescence .

How does phosphorylation at Ser1417 alter Girdin's subcellular localization and function?

Ser1417 phosphorylation by AKT induces critical changes in Girdin:

• Subcellular relocalization: Upon phosphorylation at Ser1417, Girdin relocates from cytosol to the cell membrane, particularly to lamellipodia .

• Functional consequences:

  • Enables crosslinking of actin filaments

  • Promotes formation of membrane protrusions

  • Enhances cell migration

  • Mediates cytoskeletal reorganization

• Pathway dependencies:

  • EGF stimulation promotes membrane localization through PI3K-dependent mechanisms

  • Tyrosine phosphorylation is required for AKT1-dependent phosphorylation of Ser1417

  • PI4P binding site (1390-1408) near the Ser1417 site mediates interactions with plasma membrane

This phosphorylation represents a critical regulatory mechanism by which growth factor signaling through AKT controls cell motility and morphology.

What are the implications of CCDC88A mutations in neurological disorders?

CCDC88A mutations have been identified as causative for a rare form of epileptic encephalopathy with profound clinical impacts:

• Clinical phenotype (PEHO-like syndrome):

  • Microcephaly (often progressive and postnatal)

  • Neonatal hypotonia

  • Severe seizures/epilepsy

  • Profound developmental delay

  • Face and limb edema

  • Distinctive dysmorphic features

  • Poor visual responsiveness

  • Brain malformations

• Neuroimaging findings:

  • Abnormal cortical gyration

  • Hypogenesis of corpus callosum

  • Colpocephaly

  • Reduced white matter

  • Hypoplastic vermis and brain stem

  • Subcortical band heterotopia

• Genetic basis:

  • Initially identified in three consanguineous families

  • Both homozygous variants (in consanguineous families) and compound heterozygous variants (in non-consanguineous families) have been reported

  • All reported pathogenic variants have been loss-of-function mutations

• Research implications: CCDC88A's role in neurological development suggests its critical function in:

  • Neuronal migration

  • Brain cortical development

  • Actin cytoskeleton maintenance during neurodevelopment

How does CCDC88A contribute to cell protrusion formation in cancer cells?

CCDC88A plays a crucial role in the formation of cell protrusions, which are essential for cell migration and cancer invasion:

• Experimental observations:

  • CCDC88A knockdown significantly decreased peripheral actin structures in S2-013 and PANC-1 pancreatic cancer cells

  • Fibronectin-mediated formation of membrane protrusions was inhibited upon CCDC88A knockdown

  • CCDC88A-rescue constructs abrogated these effects, confirming specificity

• Localization pattern:

  • Exogenous CCDC88A strongly accumulates in cell protrusions

  • Co-localizes with actin filaments in these protrusions

  • This localization is critical for protrusion formation and function

• Mechanistic pathway:

  • CCDC88A mediates peripheral actin structure formation

  • Facilitates membrane ruffling

  • Enables protrusive activity at the leading edge of migrating cells

  • Acts as a scaffold for signaling molecules involved in cytoskeletal organization

These findings highlight CCDC88A as a potential therapeutic target in cancers where cell migration and invasion contribute to disease progression.

What is the relationship between CCDC88A and Akt signaling?

The CCDC88A-Akt relationship represents a bidirectional regulatory system:

• Akt regulation of CCDC88A:

  • Akt (AKT1) directly phosphorylates CCDC88A at Ser1417

  • This phosphorylation is PI3K-dependent and induced by growth factors like EGF

  • Phosphorylation at Ser1417 is necessary for delocalization from cell membrane and for cell migration

• CCDC88A influence on Akt pathway:

  • CCDC88A can be tyrosine-phosphorylated by both receptor and non-receptor tyrosine kinases

  • Tyrosine phosphorylation promotes binding to PI3K regulatory subunit PIK3R1/p85a

  • This binding enhances PI3K activity, which in turn can activate Akt

  • Tyrosine phosphorylation is required for AKT1-dependent phosphorylation of Ser1417, creating a positive feedback loop

• Functional consequences:

  • This reciprocal regulation creates a sophisticated control system for cell migration

  • The CCDC88A-Akt axis represents a potential therapeutic target in cancer

  • Understanding this relationship has implications for both cancer research and neurological disorders

What are the optimal sample preparation techniques for detecting phosphorylated CCDC88A?

Detecting phosphorylated CCDC88A requires careful sample preparation to preserve phosphorylation status:

• Cell lysis buffer composition:

  • 20 mM HEPES (pH 7.4)

  • 100 mM KCl

  • 5 mM MgCl₂

  • 0.5% Triton X-100

  • Protease inhibitor cocktail tablets

  • Phosphatase inhibitor cocktail (critical for preserving phosphorylation)

• Stimulation protocols:

  • Serum starvation (16-24h) before stimulation to reduce basal phosphorylation

  • EGF stimulation (50-100 ng/mL, 5-15 min) to induce phosphorylation

  • Fibronectin coating (5 μg/mL) for adhesion-dependent phosphorylation studies

• Protein quantification:

  • Use BCA assay to determine protein concentration

  • Standardize loading to 1-2 μg/μL for consistent results

• Immunoprecipitation protocol:

  • Lyse cells and immunoprecipitate with Dynabeads Protein G

  • Use anti-CCDC88A antibody or mouse IgG isotype control antibody (2h, 4°C)

  • Pellet beads using a magnetic rack

  • Analyze by western blotting with phospho-specific antibody

How can researchers effectively study the functional consequences of Ser1417 phosphorylation?

To investigate the specific roles of Ser1417 phosphorylation:

• Site-directed mutagenesis:

  • Generate S1417A (phospho-deficient) mutant

  • Generate S1417D/E (phospho-mimetic) mutant

  • Compare these mutants in functional assays

• PI3K/Akt inhibition:

  • Pharmacological inhibitors (wortmannin, LY294002 for PI3K; MK2206 for Akt)

  • siRNA knockdown of Akt1

  • Monitor effects on Ser1417 phosphorylation and cellular functions

• Functional assays:

  • Cell migration (wound healing, transwell)

  • Membrane protrusion formation

  • Cytoskeletal reorganization

  • Cell invasion

  • Live cell imaging to track dynamics

• Interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Pull-down assays with phospho-mimetic vs. phospho-deficient mutants

  • Mass spectrometry to identify phosphorylation-dependent protein interactions

What are the best experimental controls when working with Phospho-CCDC88A (Ser1417) antibodies?

Rigorous controls are essential for phospho-specific antibody experiments:

• Positive controls:

  • Cell lines with known high Girdin expression (e.g., A431, H69AR, MRC-5)

  • Growth factor stimulated cells (EGF, IGF-1)

  • Cells transfected with constitutively active Akt

• Negative controls:

  • Phosphatase treatment of samples

  • PI3K/Akt inhibitor treated cells

  • siRNA knockdown of CCDC88A

  • Non-phosphorylated peptide competition

  • Blocking peptide competition

• Specificity controls:

  • Peptide competition assays using phosphorylated vs. non-phosphorylated peptides

  • S1417A mutant-expressing cells

  • Western blot with total CCDC88A antibody in parallel

• Technical controls:

  • Mouse IgG isotype control for immunoprecipitation

  • Loading controls for western blotting

  • Secondary antibody-only controls for immunofluorescence

What are promising research avenues for CCDC88A in neurodevelopmental disorders?

The discovery of CCDC88A mutations in epileptic encephalopathy opens several research opportunities:

• Genotype-phenotype correlations:

  • Compare clinical presentations across patients with different CCDC88A mutations

  • Analyze domain-specific mutations and their clinical consequences

  • Study whether particular mutations affect phosphorylation sites like Ser1417

• Animal models:

  • Develop conditional CCDC88A knockout mouse models

  • Create knock-in models with patient-specific mutations

  • Examine neuronal migration and brain development in these models

• Cellular mechanisms:

  • Investigate how CCDC88A mutations affect:

    • Neuronal migration

    • Axon guidance

    • Dendritic spine formation

    • Synaptic plasticity

  • Study the role of Ser1417 phosphorylation in these processes

• Therapeutic targets:

  • Explore downstream effectors that might be amenable to therapeutic intervention

  • Test whether modulating Akt-CCDC88A signaling can ameliorate pathological phenotypes

  • Investigate potential gene therapy approaches

How might targeting CCDC88A phosphorylation impact cancer treatment strategies?

Given CCDC88A's role in cancer cell migration and invasion, targeting its phosphorylation presents therapeutic potential:

• Biomarker development:

  • Evaluate Phospho-CCDC88A (Ser1417) as a prognostic marker in various cancers

  • Determine if CCDC88A phosphorylation correlates with invasiveness, metastasis, or treatment response

  • Develop tissue-based or liquid biopsy assays for clinical use

• Combination therapies:

  • Test PI3K/Akt inhibitors in combination with other targeted therapies

  • Explore synergistic effects with cytoskeletal-targeting drugs

  • Investigate whether CCDC88A status affects response to standard chemotherapies

• Novel therapeutic approaches:

  • Develop peptide inhibitors targeting the Ser1417 region

  • Screen for small molecules that specifically block CCDC88A phosphorylation

  • Explore nanoparticle-based delivery of anti-CCDC88A siRNAs to tumors

• Resistance mechanisms:

  • Study whether CCDC88A phosphorylation contributes to treatment resistance

  • Identify alternative pathways that compensate for CCDC88A inhibition

  • Develop strategies to overcome potential resistance mechanisms