C14ORF129 Human

What is C14ORF129 and what are its alternative names?

C14ORF129 is officially known as GSK3-beta interaction protein (GSKIP). Additional synonyms include HSPC210, MGC4945, and HSPC120. The name C14orf129 derives from its genomic location as an open reading frame on chromosome 14. The protein is defined as a naturally occurring protein homologous with the GSK3beta interaction domain of Axin that negatively regulates GSK3beta in the Wnt signaling pathway . The human GSKIP gene encodes four alternatively spliced transcripts, with splice variant 1 being the longest; the others differ only in their 5′-UTR compared to transcript 1 .

What is the molecular structure and characteristics of C14ORF129?

C14ORF129 Human Recombinant protein contains 159 amino acids (including a 20 amino acid His tag at N-terminus) with the core protein being 139 amino acids in length. When produced recombinantly in E.coli, it forms a single, non-glycosylated polypeptide chain with a molecular mass of approximately 17.8 kDa . The protein structure includes a PKA-binding domain encoded by exon 2 of the gene, which constitutes 86 of the total 139 amino acids . This structural feature is critical for its function as an A-kinase anchoring protein (AKAP) that interacts specifically with the regulatory RII subunits of protein kinase A (PKA) .

Where is C14ORF129/GSKIP primarily expressed in human tissues?

While the search results don't provide comprehensive information about tissue-specific expression patterns of C14ORF129/GSKIP in humans, developmental studies in mice offer insights into its expression profile. GSKIP expression has been detected across multiple embryonic tissues at various developmental stages from E10.5 to E18.5 . The protein appears to be developmentally regulated, with significant functions in craniofacial development. The location of the GSKIP gene in a chromosomal region linked to hemifacial microsomia (Goldenhar syndrome) suggests important roles in facial tissue development . Additionally, duplication of the chromosomal region containing GSKIP (14q32.13-q32.2) has been associated with myeloid malignancies, indicating potential roles in hematopoietic tissues .

How does C14ORF129/GSKIP interact with the Wnt signaling pathway?

C14ORF129/GSKIP interacts with the Wnt signaling pathway primarily through its regulation of GSK3β, a key pathway component. GSKIP shares homology with the GSK3beta interaction domain of Axin, a major scaffolding protein in the Wnt pathway . Through this interaction, GSKIP negatively regulates GSK3β activity . In canonical Wnt signaling, GSK3β typically phosphorylates β-catenin, targeting it for degradation. When GSK3β is inhibited, β-catenin accumulates and activates Wnt target genes. Interestingly, knockout studies revealed that despite changes in GSK3β activity with GSKIP depletion, expression levels of key Wnt pathway components including β-catenin, Axin1, and the Wnt target gene cyclin D1 remained unaltered . This suggests GSKIP's regulation of GSK3β may function in specific cellular contexts or affect particular GSK3β pools rather than globally influencing canonical Wnt signaling.

How do Ser-9 phosphorylation levels of GSK3β change in the absence of GSKIP?

The absence of GSKIP has significant effects on GSK3β phosphorylation status, but these effects vary by developmental stage. Research using knockout mouse models revealed:

This data demonstrates that GSKIP influences GSK3β through development-specific mechanisms, with a dramatic shift occurring between early and late embryonic stages .

What protein detection methods are most effective for studying C14ORF129/GSKIP?

Based on published research, several effective methods have been established for C14ORF129/GSKIP detection:

• Western Blot Analysis: This primary technique for GSKIP protein detection utilizes:

  • Custom-made GSKIP antibodies (previously validated)

  • Standard radioimmunoprecipitation assay buffer with protease/phosphatase inhibitors

  • Protein separation via 12% SDS-PAGE and transfer to PVDF membranes

  • Blocking with 1% BSA in TBS-T

  • Visualization using chemiluminescence detection systems

• Semi-quantitative Analysis: Using software such as ImageJ for quantification of Western blot signals, with normalization to housekeeping proteins like GAPDH

• Activity Assessment: For functional studies, measuring GSK3β phosphorylation status (Ser-9) as a readout of GSKIP activity, calculating the ratio between phosphorylated (inactive) and total GSK3β

For comprehensive analysis, researchers should combine protein detection with mRNA quantification (qPCR) to correlate transcript and protein levels, especially when studying splice variants or developmental expression patterns.

How can researchers generate reliable knockout models for GSKIP studies?

The search results describe a validated approach to generating GSKIP knockout models using the Cre/loxP system . This methodology involves:

• Strategic Targeting: Flanking exon 2 of the GSKIP gene (containing the start codon and PKA-binding domain) with loxP sites for Cre-mediated deletion

• Comprehensive Validation at Multiple Levels:

  • DNA level: PCR-based genotyping

  • mRNA level: qPCR to confirm transcript elimination

  • Protein level: Western blotting to verify complete protein depletion

• Conditional Approach: Since complete GSKIP knockout causes perinatal lethality, conditional systems allow for temporal and tissue-specific deletion studies

For cellular models, CRISPR-Cas9 technology targeting similar regions of the GSKIP gene could complement animal models for mechanistic studies.

What controls are essential when analyzing GSK3β activity in GSKIP research?

When investigating GSK3β activity in relation to GSKIP function, researchers should implement several critical controls:

• Phosphorylation Status Controls:

  • Always measure both phosphorylated (Ser-9) and total GSK3β protein

  • Calculate the ratio of inactive (phosphorylated) to total GSK3β as the primary activity metric

  • Include positive controls for phosphorylation (e.g., insulin-treated samples)

• Genotype Controls:

  • Include wild-type, heterozygous, and homozygous knockout samples to establish dose-dependent effects

  • Use littermate controls to minimize genetic background variation

• Developmental Stage Controls:

  • Analyze multiple developmental timepoints, as GSKIP's effects on GSK3β activity change significantly between early (E10.5-E14.5) and late (E18.5) embryonic stages

  • Create developmental timelines rather than single-point analyses

• Pathway Validation:

  • Examine downstream targets of GSK3β (e.g., β-catenin, cyclin D1) to confirm functional consequences of activity changes

  • Include positive controls for pathway activation/inhibition

• Technical Controls:

  • Use loading controls (GAPDH) for Western blot normalization

  • Implement appropriate negative controls for antibody specificity

These controls ensure accurate interpretation of GSK3β activity data and its relationship to GSKIP function.

How might C14ORF129/GSKIP function in neurodevelopment?

GSKIP likely plays important roles in neurodevelopment through its regulation of GSK3β, although detailed neural functions remain to be fully characterized. Current research indicates that GSKIP "may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396)" . This suggests potential functions in neuronal morphogenesis and axonal development. GSK3β is a well-established regulator of tau phosphorylation, microtubule dynamics, and neuronal polarity, making GSKIP's regulatory role potentially significant for multiple aspects of neural development and function. The severe developmental phenotypes in GSKIP knockout mice, including craniofacial abnormalities and perinatal lethality , could involve neural components, particularly in craniofacial development where neural crest cells play critical roles. Future research directions should include:

• Neural-specific conditional knockout models

• Analysis of GSKIP expression in developing neural tissues

• Investigation of neurite outgrowth in GSKIP-depleted neuronal cultures

• Examination of tau phosphorylation patterns in neural tissues lacking GSKIP

These approaches would help elucidate GSKIP's specific contributions to neurodevelopmental processes.

What can GSKIP knockout phenotypes tell us about potential human disorders?

GSKIP knockout studies provide valuable insights into potential human disorders associated with GSKIP dysfunction:

• Craniofacial Disorders: GSKIP knockout mice develop cleft palate and die perinatally due to respiratory distress . The human GSKIP gene is located at 14q32, a region linked to hemifacial microsomia (Goldenhar syndrome) through genome-wide linkage studies . This syndrome presents with craniofacial abnormalities affecting the ear, mandible, muscle, and facial soft tissues, sometimes with tracheal obstruction and breathing difficulties - remarkably similar to the mouse phenotype.

• Developmental Disorders: The perinatal lethality of knockout mice indicates GSKIP's essential role in developmental processes , suggesting that human GSKIP mutations might be embryonically lethal or cause severe developmental abnormalities.

• Hematological Malignancies: Duplication of the chromosomal region containing GSKIP (14q32.13-q32.2) has been associated with myeloid malignancies , indicating that increased GSKIP expression might contribute to certain cancers.

• Wnt Signaling Disorders: As GSKIP regulates GSK3β in the Wnt pathway , dysfunction might impact the many developmental and homeostatic processes controlled by this pathway, potentially contributing to conditions associated with aberrant Wnt signaling.

These correlations suggest that GSKIP should be considered a candidate gene in human developmental disorders, particularly those affecting craniofacial development.

What experimental design factors contribute to inconsistent results in GSK3β-related research?

Research on GSK3β regulation often produces inconsistent results, similar to challenges seen in other fields like C-bouton research in ALS . Several experimental design factors can contribute to these inconsistencies:

• Sampling Strategy Variations:

  • Inconsistent tissue sampling methods

  • Selection of "representative" fields versus systematic sampling

  • Variation in anatomical regions examined

• Grouping Unit Decisions:

  • Using animals versus sections versus cells as the statistical unit

  • Pooling samples across different developmental stages

  • Combining data from different genetic backgrounds

• Blinding Status:

  • Unblinded analysis increasing risk of confirmation bias

  • Inconsistent blinding practices across studies

  • Inadequate reporting of blinding methodologies

• Developmental Timing:

  • GSKIP's effects on GSK3β change dramatically between developmental stages

  • Inconsistent age-matching between studies

  • Failure to account for dynamic developmental processes

• Quantification Methods:

  • Variation in image analysis software and settings

  • Inconsistent normalization approaches

  • Different methods for calculating activity ratios

To overcome these challenges, researchers should implement standardized protocols, thorough methodological reporting, and robust statistical approaches.

How should researchers design experiments to resolve conflicting data about GSKIP function?

To resolve conflicting findings regarding GSKIP function, researchers should implement a systematic experimental approach:

• Standardized Protocol Development:

  • Establish consistent tissue preparation methods

  • Standardize protein extraction and detection protocols

  • Use validated antibodies with documented specificity

  • Implement uniform activity assays for GSK3β

• Comprehensive Developmental Analysis:

  • Examine multiple precisely-defined developmental stages

  • Create continuous developmental timelines rather than isolated timepoints

  • Account for the dramatic shift in GSKIP-GSK3β relationship between early and late development

• Multi-level Validation:

  • Verify findings at DNA, RNA, and protein levels

  • Confirm functional consequences through downstream target analysis

  • Utilize multiple experimental approaches to address the same question

• Rigorous Controls and Blinding:

  • Implement strict blinding procedures for all analysis steps

  • Include appropriate positive and negative controls

  • Use littermate controls to minimize genetic background effects

• Statistical Rigor:

  • Conduct power analyses to determine appropriate sample sizes

  • Pre-specify primary outcomes and analysis methods

  • Apply appropriate statistical tests and corrections for multiple comparisons

This methodological framework, combined with transparent reporting of all procedures, will help resolve discrepancies and establish consistent understanding of GSKIP function.

What statistical considerations are critical when analyzing GSK3β phosphorylation in GSKIP studies?

Statistical analysis of GSK3β phosphorylation in GSKIP research requires careful consideration of several factors:

These statistical approaches enhance the reliability and interpretability of GSK3β phosphorylation data in GSKIP research.

What reporting standards should researchers follow when publishing GSKIP-related findings?

Based on methodological considerations highlighted in the search results , researchers should adhere to the following reporting standards when publishing GSKIP-related findings:

Following these reporting standards will enhance reproducibility and facilitate resolution of conflicting findings in GSKIP research.