LAMTOR4 Human

What is LAMTOR4 and what is its primary function in human cells?

LAMTOR4, also known as C7orf59 (Chromosome 7 Open Reading Frame 59), is a component of the pentameric Ragulator complex that plays a crucial role in amino acid sensing and activation of mTORC1 signaling. This 99-amino acid protein functions as part of a complex that promotes cell growth in response to growth factors, energy levels, and amino acids . LAMTOR4 serves as a nucleating factor for p18 (LAMTOR1) and acts as a phosphorylation target within the Ragulator complex . The protein is involved in mediating the activation of the mTORC1 pathway, which regulates numerous cellular processes including protein synthesis, cell growth, and metabolism.

How does LAMTOR4 differ structurally from other LAMTOR proteins?

LAMTOR4 features a unique structural profile compared to other members of the LAMTOR family. Crystal structure analysis at 2.9 Å resolution reveals that LAMTOR4 contains an unfolded N-terminus (residues 1-15) that is essential for p18 binding . While LAMTOR4 forms a heterodimer with LAMTOR5 (HBXIP), it exhibits distinct functional characteristics. The protein contains approximately 99 amino acids and forms a roadblock domain that facilitates protein-protein interactions within the Ragulator complex . Unlike LAMTOR1 (p18), which contains a lipid anchor, LAMTOR4 relies on protein-protein interactions for its localization and function.

What is the evolutionary conservation of LAMTOR4 across species?

LAMTOR4 shows evolutionary relationships with components of the yeast Ego complex, specifically with Ego2/Ycr075w-a, although the relationship is not as clearly defined as other LAMTOR proteins . The functional conservation of LAMTOR4 across species reflects its fundamental role in nutrient sensing pathways that have been preserved throughout eukaryotic evolution. While the mammalian Ragulator complex and yeast Ego complex share functional and structural features, such as lysosomal/vacuolar localization and interaction with Rag GTPases, the evolutionary relationships between specific subunits are sometimes ambiguous . This evolutionary conservation underscores the importance of LAMTOR4 in cellular function and suggests it emerged as part of ancient nutrient-sensing mechanisms.

How does LAMTOR4 contribute to Ragulator complex assembly and stability?

LAMTOR4 serves as a critical nucleating factor for Ragulator complex assembly, particularly by binding and stabilizing p18 (LAMTOR1) . Research has demonstrated that LAMTOR4 forms a stable heterodimer with LAMTOR5 (HBXIP), and this dimer is essential for the initial steps of Ragulator assembly. The LAMTOR4-LAMTOR5 dimer binds and stabilizes p18, which subsequently allows the binding of the MP1-p14 dimer (LAMTOR2-LAMTOR3) . This sequential assembly model highlights LAMTOR4's role as a foundational component for proper Ragulator complex formation.

The unfolded N-terminus of LAMTOR4 (residues 1-15) has been shown to be essential for p18 binding, serving as a flexible interaction interface . When this region is deleted or mutated, the ability of LAMTOR4 to interact with p18 is significantly compromised, affecting the entire Ragulator assembly process. This demonstrates that LAMTOR4 not only participates in the complex but plays an architectural role in establishing its structure.

What is the mechanism by which LAMTOR4 affects mTORC1 activation?

LAMTOR4 influences mTORC1 activation through multiple mechanisms within the amino acid sensing pathway. As part of the Ragulator complex, LAMTOR4 contributes to the guanine nucleotide exchange factor (GEF) activity that activates Rag GTPases in response to amino acids . This activation occurs through a mechanism involving the lysosomal V-ATPase, where the Ragulator complex:

• Acts as a GEF to activate small GTPases Rag (RagA/RRAGA, RagB/RRAGB, RagC/RRAGC, and/or RagD/RRAGD)

• Mediates recruitment of Rag GTPases to the lysosomal membrane

• Creates a scaffold that recruits mTORC1 to lysosomes, where it becomes activated

Quantitative genetic screening has revealed that LAMTOR4 has distinct effects on mTORC1 signaling compared to LAMTOR5, particularly in enhancing the inhibition of mTORC1 by rapamycin, despite both proteins being part of the same complex . This suggests LAMTOR4 may have additional regulatory functions beyond its structural role in Ragulator assembly.

How does the interaction between LAMTOR4 and FLCN affect cellular signaling pathways?

LAMTOR4 has demonstrated a synthetic sick/lethal interaction with folliculin (FLCN), suggesting important functional relationships between these proteins in cellular signaling . While both proteins participate in nutrient sensing pathways, their interaction points to potential co-regulation or interdependence in controlling mTORC1 activation. Genetic screening approaches have shown that LAMTOR4 loss creates cellular vulnerabilities that are exacerbated when FLCN function is also compromised .

This interaction is particularly significant because FLCN is a tumor suppressor gene, mutations in which are associated with Birt-Hogg-Dubé syndrome. The LAMTOR4-FLCN relationship suggests potential crosstalk between the Ragulator complex and FLCN-mediated tumor suppression pathways, which could have implications for understanding certain cancer mechanisms and developing targeted therapies.

What are the most effective methods for studying LAMTOR4 function in cell culture systems?

For investigating LAMTOR4 function in cell culture systems, several approaches have demonstrated particular effectiveness:

• CRISPR-Cas9 gene editing: Creating LAMTOR4 knockout cell lines using CRISPR-Cas9 technology has proven valuable for assessing its function. The HAP1 haploid human cell line has shown reproducible results in generating LAMTOR4-deficient cells across different laboratories without explicit standardization .

• Phosphorylation state analysis: Using phospho-specific antibodies against downstream targets like phosphorylated ribosomal protein S6 (p-RPS6) provides a quantitative readout of mTORC1 activity in response to LAMTOR4 manipulation .

• Co-immunoprecipitation assays: These assays are effective for studying LAMTOR4's interactions with other Ragulator components and identifying novel binding partners. They can reveal how LAMTOR4 participates in protein complex formation under various cellular conditions .

• PKA modulation: Using forskolin to activate PKA and H-89 to inhibit it has proven useful for studying the phosphorylation-dependent regulation of LAMTOR4 and its interactions with p18 .

• Subcellular localization studies: Immunofluorescence microscopy with lysosomal markers helps determine how LAMTOR4 manipulation affects Ragulator complex assembly and mTORC1 recruitment to lysosomes .

What are the challenges in expressing and purifying recombinant LAMTOR4 for structural studies?

Researchers face several challenges when expressing and purifying recombinant LAMTOR4 for structural studies:

• Solubility issues: LAMTOR4's natural tendency to form a heterodimer with LAMTOR5 means that expressing it alone may lead to solubility problems or improper folding. Co-expression with LAMTOR5 is often necessary to obtain properly folded protein .

• Flexible regions: The unfolded N-terminus of LAMTOR4 (residues 1-15) that is critical for p18 binding can create challenges for crystallization due to its inherent flexibility .

• Expression system selection: While E. coli expression systems can produce LAMTOR4 (as evidenced by commercially available recombinant proteins), obtaining functional protein requires careful optimization of expression conditions .

• Protein stability: Maintaining LAMTOR4 stability during purification requires specific buffer conditions, including the addition of stabilizing agents like glycerol and reducing agents such as DTT .

• Purity requirements: For structural studies, high purity (>85%) is essential, requiring multiple chromatographic steps and careful quality control .

For successful purification, protocols typically include His-tag affinity chromatography followed by additional purification steps, with storage recommendations including glycerol addition and avoidance of multiple freeze-thaw cycles .

What genetic screening approaches are most informative for studying LAMTOR4 in the context of mTORC1 signaling?

Quantitative genetic screening approaches have proven particularly valuable for investigating LAMTOR4's role in mTORC1 signaling:

• Haploid genetic screens: Studies in HAP1, a near-haploid human cell line, have produced reproducible results across different laboratories without requiring explicit standardization. This approach has successfully identified genes that, when mutated, affect phosphorylation of RPS6 (a measure of mTORC1 activity) .

• Combined pharmacological and genetic perturbations: Adding rapamycin to genetic screens has revealed that LAMTOR4 enhances the inhibition of mTORC1 by this drug, an effect not demonstrated by the related LAMTOR5. This approach helps identify context-specific functions of Ragulator components .

• Synthetic lethality screening: This approach has identified a synthetic sick/lethal interaction between LAMTOR4 and folliculin, revealing important functional connections that might not be apparent through single-gene studies .

• Site-directed mutagenesis: Targeted mutation of specific residues, such as the Ser67 phosphorylation site in LAMTOR4, combined with functional readouts, has elucidated regulatory mechanisms controlling Ragulator assembly and function .

• Perturb-and-observe approach: This methodology, which systematically measures cellular responses to genetic perturbations, has helped reveal signaling pathway regulators that function in specific contexts, including LAMTOR4's role in mTORC1 regulation .

How does phosphorylation of LAMTOR4 affect its interaction with other Ragulator components?

Phosphorylation of LAMTOR4 plays a crucial regulatory role in modulating its interactions within the Ragulator complex. Research has identified Ser67 as a key phosphorylation site that significantly affects LAMTOR4's binding capacity with p18 (LAMTOR1) . Experimental evidence shows that:

• Mutation of the conserved Ser67 residue to aspartate (phosphomimetic mutation) prevented further phosphorylation and negatively affected LAMTOR4's interaction with p18 both in cell culture and in vitro studies .

• Ser67 was confirmed to be phosphorylated in human embryonic kidney 293T cells, establishing the physiological relevance of this modification .

• PKA activation with forskolin induced dissociation of p18 from LAMTOR4, demonstrating that phosphorylation can dynamically regulate complex stability .

• The PKA inhibitor H-89 prevented this dissociation, further confirming the role of PKA-mediated phosphorylation in regulating LAMTOR4-p18 interactions .

These findings indicate that phosphorylation serves as a molecular switch that can modulate Ragulator complex assembly and potentially fine-tune mTORC1 signaling in response to changing cellular conditions.

What kinases and phosphatases regulate LAMTOR4 activity?

Research has identified Protein Kinase A (PKA) as a significant regulator of LAMTOR4 through phosphorylation at Ser67 . Experimental evidence supports PKA's role:

• PKA activation effects: Treatment with forskolin, a PKA activator, induces dissociation of p18 from LAMTOR4, suggesting PKA-mediated phosphorylation destabilizes this interaction .

• PKA inhibition effects: The specific PKA inhibitor H-89 prevents the forskolin-induced dissociation of p18 from LAMTOR4, confirming PKA's regulatory role .

While PKA has been established as a key kinase regulating LAMTOR4, the specific phosphatases responsible for dephosphorylating LAMTOR4 and potentially re-establishing Ragulator complex stability have not been definitively identified in the available research. This represents an important area for future investigation, as phosphatases would complete the regulatory circuit controlling LAMTOR4's phosphorylation state.

The identification of PKA as a LAMTOR4 regulator is particularly significant because it suggests a mechanism for integrating cAMP signaling with amino acid sensing and mTORC1 activation, potentially linking diverse cellular signaling networks through LAMTOR4 phosphorylation.

What is the significance of LAMTOR4's unfolded N-terminus in protein-protein interactions?

The unfolded N-terminus of LAMTOR4 (residues 1-15) serves as a critical structural element mediating specific protein-protein interactions within the Ragulator complex . Research findings demonstrate:

• This unfolded region is essential for p18 binding, as demonstrated through structural and functional studies .

• The flexibility of this N-terminal segment likely provides adaptability in binding, allowing LAMTOR4 to accommodate conformational changes during Ragulator assembly.

• This structural feature distinguishes LAMTOR4 from other Ragulator components and contributes to its specific role as a nucleator for complex assembly.

The presence of this disordered region highlights the importance of intrinsically disordered protein segments in mediating dynamic protein interactions. Such regions often serve as molecular recognition elements that can adopt different conformations when binding to partners, providing both specificity and adaptability in protein complex formation.

The dual presence of structured domains and unstructured regions in LAMTOR4 exemplifies a common theme in signaling proteins, where ordered regions provide stable structural scaffolds while disordered segments enable dynamic and regulated interactions with binding partners.

How might targeting LAMTOR4 be exploited therapeutically in diseases with dysregulated mTORC1 signaling?

Targeting LAMTOR4 presents a novel therapeutic strategy for diseases characterized by dysregulated mTORC1 signaling, including certain cancers, diabetes, and neurodegenerative disorders . Several approaches warrant investigation:

• Enhancing rapamycin sensitivity: LAMTOR4 has been shown to enhance the inhibition of mTORC1 by rapamycin, an effect not demonstrated by LAMTOR5 . This unique property could be exploited to develop combination therapies that specifically target LAMTOR4 to sensitize cancer cells to rapalogs (rapamycin analogs), potentially overcoming resistance mechanisms.

• Disrupting protein-protein interactions: The interaction between LAMTOR4 and p18, mediated by LAMTOR4's unfolded N-terminus, represents a potential target for small molecule or peptide-based inhibitors . Disrupting this interaction could prevent proper Ragulator assembly and subsequent mTORC1 activation.

• Modulating phosphorylation: Given that phosphorylation of LAMTOR4 at Ser67 affects its interaction with p18, developing compounds that mimic or enhance this phosphorylation could provide a mechanism to regulate Ragulator assembly and mTORC1 activity .

• Exploiting synthetic lethality: The synthetic sick/lethal interaction between LAMTOR4 and folliculin (FLCN) suggests potential for developing targeted therapies for specific genetic backgrounds . For example, in tumors with FLCN mutations, LAMTOR4 inhibition might prove selectively toxic.

• Amino acid sensing modulation: Since LAMTOR4 participates in amino acid sensing that activates mTORC1, targeting this specific function could provide more selective inhibition of mTORC1 in response to nutrient signals while potentially preserving other mTORC1 functions .

What are the current gaps in understanding LAMTOR4's role in non-canonical signaling pathways?

Despite significant advances in understanding LAMTOR4's function within the Ragulator complex and mTORC1 pathway, several knowledge gaps exist regarding its potential roles in non-canonical signaling pathways:

• MAPK pathway involvement: While LAMTOR4 is classified as a "MAPK and mTOR activator," its specific contributions to MAPK signaling remain less characterized than its mTORC1-related functions . How LAMTOR4 might coordinate or balance inputs between MAPK and mTOR pathways deserves further investigation.

• Tissue-specific functions: The available research does not fully address whether LAMTOR4 has tissue-specific roles or if its function varies across different cell types. Understanding potential tissue-specific regulation could reveal specialized functions beyond its core role in amino acid sensing.

• Stress response pathways: Whether LAMTOR4 participates in cellular stress response pathways independent of mTORC1 signaling remains an open question. Its potential involvement in adapting to various cellular stresses beyond nutrient limitation warrants exploration.

• Developmental roles: The function of LAMTOR4 during development and its potential contributions to cell differentiation processes have not been extensively characterized in the available research.

• Interaction with other GTPases: While LAMTOR4's role in regulating Rag GTPases is established, its potential interactions with other small GTPases beyond the Ragulator complex remain largely unexplored.

What methodological innovations are needed to better understand LAMTOR4's dynamic behavior in living cells?

Advancing our understanding of LAMTOR4's dynamic behavior in living cells will require several methodological innovations:

• Live-cell imaging technologies: Development of fluorescent protein tags or biosensors that do not disrupt LAMTOR4 function would enable real-time visualization of its localization, complex formation, and response to various stimuli. This could provide insights into the temporal dynamics of Ragulator assembly and disassembly.

• Single-molecule techniques: Applying single-molecule tracking or super-resolution microscopy to LAMTOR4 would reveal its diffusion dynamics, potential membrane associations, and nanoscale organization within the lysosomal membrane.

• Proximity labeling approaches: Techniques like BioID or APEX2 proximity labeling could identify transient or context-specific LAMTOR4 interactions that might be missed in traditional co-immunoprecipitation experiments, particularly in response to different cellular conditions.

• Phosphoproteomic profiling: Comprehensive phosphoproteomic analysis of LAMTOR4 under various cellular conditions could identify additional phosphorylation sites beyond Ser67 and their regulatory significance .

• Cryo-electron microscopy: While crystal structures of LAMTOR4-LAMTOR5 dimers exist , cryo-EM could potentially reveal the structure of the entire Ragulator complex and its association with Rag GTPases and the lysosomal membrane, providing mechanistic insights into LAMTOR4's function within this larger assembly.

• Optogenetic tools: Development of optogenetic approaches to rapidly modulate LAMTOR4 phosphorylation or interactions would enable precise temporal control over its function, helping to dissect the kinetics of Ragulator assembly and mTORC1 activation.

How do the functional differences between LAMTOR4 and LAMTOR5 impact experimental design?

Despite being partners in the same heterodimer, LAMTOR4 and LAMTOR5 exhibit distinct functional characteristics that significantly impact experimental design:

These differences necessitate careful experimental design decisions, including:

• Individual knockout/knockdown studies rather than simultaneous depletion

• Separate analysis of downstream signaling effects

• Consideration of context-dependent functions in different experimental systems

• Inclusion of phosphorylation state analysis when studying LAMTOR4 specifically

What are the most reliable cellular models for studying LAMTOR4 function in different disease contexts?

Several cellular models have proven valuable for studying LAMTOR4 function across different disease contexts:

• HAP1 cells: This near-haploid human cell line has generated reproducible results in studying LAMTOR4 function across different laboratories without explicit standardization . The haploid nature simplifies genetic manipulation and phenotypic analysis, making it ideal for genetic screening approaches.

• HEK293T cells: Human embryonic kidney cells have been successfully used to study LAMTOR4 phosphorylation, demonstrating that Ser67 is phosphorylated under physiological conditions . These cells are particularly useful for protein overexpression studies and examining LAMTOR4 regulation.

• Cancer cell lines with mTORC1 hyperactivation: Various cancer cell lines with constitutive mTORC1 activation represent valuable models for studying how LAMTOR4 manipulation might affect tumor cell growth and survival. These include TSC1/2-deficient cell lines that model tuberous sclerosis complex.

• Nutrient-responsive cell types: Cell types highly responsive to nutrient fluctuations, such as pancreatic β-cells, hepatocytes, and myocytes, may be particularly informative for studying LAMTOR4's role in metabolic diseases, as these cells rely heavily on proper nutrient sensing via the mTORC1 pathway.

• Neuronal models: Given the importance of mTORC1 signaling in neurodegenerative diseases, neuronal cell models may help elucidate LAMTOR4's potential role in conditions like Alzheimer's and Parkinson's diseases, where aberrant mTORC1 signaling has been implicated.

What quality control measures are essential when working with recombinant LAMTOR4 in biochemical assays?

When working with recombinant LAMTOR4 in biochemical assays, several critical quality control measures should be implemented:

• Purity assessment: SDS-PAGE analysis should confirm >85% purity, as lower purity can introduce confounding factors in interaction studies or enzymatic assays .

• Proper folding verification: Circular dichroism spectroscopy can help verify that recombinant LAMTOR4 maintains its proper secondary structure, particularly important given the presence of both structured domains and unstructured regions.

• Functional validation: Confirming LAMTOR4's ability to interact with known binding partners (particularly LAMTOR5 and LAMTOR1) through pull-down assays provides critical validation of functional integrity .

• Stability monitoring: Thermal shift assays can assess protein stability under various buffer conditions, helping optimize storage and experimental conditions to prevent degradation.

• Phosphorylation state control: When studying phosphorylation-dependent interactions, mass spectrometry should verify the phosphorylation state of key residues like Ser67 .

• Storage condition optimization: Recombinant LAMTOR4 requires specific storage conditions, including addition of carrier proteins (0.1% HSA or BSA) for long-term storage, glycerol addition, and avoidance of multiple freeze-thaw cycles .

• Co-purification consideration: For many applications, co-expression and co-purification with LAMTOR5 may be necessary to maintain proper folding and function, as these proteins naturally exist as a heterodimer .