RHEBL1 Antibody

What is RHEBL1 and what cellular functions does it regulate?

RHEBL1 (ras homolog enriched in brain-like protein 1) is a 183 amino acid GTPase protein that plays crucial roles in cellular signaling pathways. It functions primarily through regulating the activity of Rictor, a key component of the mTOR signaling pathway . RHEBL1 is involved in numerous cellular processes including growth, proliferation, and survival. Importantly, RHEBL1 has been shown to bind and activate AKT1, demonstrating its significance in signal transduction . The protein is localized to both cell membrane and cytoplasm, which is essential for its function in signaling cascades. RHEBL1's expression is often upregulated in tumor cell lines, suggesting its importance in cancer biology .

Unlike its paralog RHEB2, RHEBL1 is critical for embryonic survival as evidenced by embryonic lethality between E10.5 and E11.5 in germline RHEBL1 deletion models . This demonstrates that despite structural similarities between RHEBL1 and RHEB2, they possess distinct physiological roles with RHEBL1 being essential for embryonic development.

How do researchers differentiate between RHEBL1 and RHEB2 in experimental systems?

Researchers can differentiate between RHEBL1 and RHEB2 through several methodological approaches. First, using specific antibodies that recognize unique epitopes of each protein is essential. The RHEBL1 Antibody (A-2), a mouse monoclonal IgG1 antibody, specifically detects RHEBL1 in human samples without cross-reactivity to RHEB2 .

Second, functional differentiation can be assessed through knockout studies. Research demonstrates that germline deletion of RHEBL1 results in embryonic death between E10.5 and E11.5, while RHEB2 knockout mice develop normally, mature to adulthood without obvious physical deficits, and are fertile . This striking difference in phenotype provides a clear functional distinction between these paralogs.

Third, signaling pathway analysis reveals that RHEBL1, but not RHEB2, is essential for mTORC1 signaling. In mouse embryonic fibroblasts (MEFs) from RHEBL1-knockout mice, insulin failed to induce phosphorylation of S6 kinase and S6, despite the presence of RHEB2, indicating RHEBL1's unique role in mTORC1 activation . These approaches collectively enable researchers to effectively distinguish between these related proteins in their experimental systems.

What are the optimal applications for RHEBL1 antibody in cancer research?

Immunoprecipitation with RHEBL1 antibody enables researchers to study protein-protein interactions, particularly with AKT1, which is crucial for understanding RHEBL1's role in cancer progression. Co-immunoprecipitation experiments revealed that RHEBL1 binds to AKT1, and this binding is dependent on RHEBL1's G protein activity .

Immunofluorescence applications allow visualization of RHEBL1 cellular localization and its co-localization with keratin filaments. Confocal microscopy using RHEBL1 antibody showed that in A549 lung cancer cells, RHEBL1 localization patterns were similar to keratin filaments, and both reorganized to perinuclear ring-like structures upon sphingosylphosphorylcholine (SPC) treatment .

For studying the functional consequences of RHEBL1 expression, researchers can employ gene silencing or overexpression approaches followed by RHEBL1 antibody detection to confirm manipulation success. This methodology revealed that RHEBL1 overexpression induced phosphorylation of K8 in multiple lung cancer cell lines (A549, H1299, H1703, and H838) and promoted migration and invasion in A549 cells .

How can researchers effectively validate RHEBL1 antibody specificity in their experimental systems?

Validating RHEBL1 antibody specificity requires a multi-faceted approach. First, researchers should employ gene knockout or knockdown controls. RHEBL1 antibody should show significantly reduced or absent signal in RHEBL1-knockout or knockdown samples, as demonstrated in various studies where RHEBL1 protein was undetectable in RHEBL1-knockout MEFs .

Multiple antibody validation is recommended, where researchers use at least two different antibodies targeting different epitopes of RHEBL1 and compare the detection patterns. Consistent results between different antibodies increase confidence in specificity.

Recombinant protein competition assays can be performed, where pre-incubation of the antibody with purified RHEBL1 protein should eliminate or significantly reduce the signal in subsequent applications if the antibody is specific. Additionally, tissue-specific expression patterns should be confirmed by comparing antibody detection with known RHEBL1 mRNA expression profiles across tissues.

For advanced validation, researchers can perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down RHEBL1 and to identify any cross-reactive proteins. This comprehensive validation approach ensures reliable results in RHEBL1 research applications.

How does RHEBL1 regulate AKT1 activation and what are the downstream effects?

RHEBL1 directly binds to and activates AKT1, serving as an upstream regulator in the AKT signaling pathway. Co-immunoprecipitation experiments confirmed that RHEBL1 interacts with AKT1, and this interaction was enhanced upon sphingosylphosphorylcholine (SPC) treatment in A549 lung cancer cells . Importantly, this binding is dependent on RHEBL1's G protein activity, as demonstrated by experiments using constitutively active (RHEBL1Q64L) and dominant negative (RHEBL1D60K) mutants .

RHEBL1 promotes phosphorylation of AKT1 at two critical residues: T308 and S473. SPC-induced phosphorylation of these residues was inhibited by gene silencing of RHEBL1, while overexpression of RHEBL1 induced phosphorylation even without SPC treatment . The constitutively active RHEBL1Q64L also induced AKT1 phosphorylation, whereas the dominant negative RHEBL1D60K did not, confirming the importance of RHEBL1's G protein activity in AKT1 activation .

The downstream effects of RHEBL1-mediated AKT1 activation include:

• Phosphorylation of keratin 8 (K8) at S431, leading to reorganization of keratin filaments into perinuclear ring-like structures

• Enhanced cancer cell migration and invasion capabilities

• Altered physical properties of metastatic cancer cells

These effects were blocked by MK2206 (an AKT inhibitor) and by gene silencing of AKT1, while overexpression of activated-AKT1 induced these phenomena even without SPC treatment . This signaling cascade represents a potential therapeutic target for preventing cancer metastasis.

What is the role of RHEBL1 in mTORC1 and mTORC2 pathway regulation?

RHEBL1 plays a critical and non-redundant role in mTORC1 signaling while also influencing mTORC2 activity through feedback mechanisms. Studies using RHEBL1-knockout mouse embryonic fibroblasts (MEFs) demonstrated that RHEBL1 is essential for mTORC1 activation. In these cells, insulin failed to induce phosphorylation of S6 kinase and S6 (indicators of mTORC1 activity), despite the presence of RHEB2, indicating that RHEBL1 specifically is required for mTORC1 activation .

Interestingly, RHEBL1 deletion affects the balance between mTORC1 and mTORC2 signaling. Embryonic deletion of RHEBL1 in neural progenitor cells abolished mTORC1 signaling in developing brain while increasing mTORC2 signaling . This relationship is mediated through a negative feedback mechanism: when RHEBL1 activates mTORC1, the subsequent activation of S6K1 inhibits mTORC2 activity through:

• Inhibition of IRS1/2 transcription

• Promotion of IRS1/2 degradation

• Direct phosphorylation of Rictor (an mTORC2 component) at T1135

Conversely, when RHEBL1 is absent, this inhibitory feedback is relieved, leading to enhanced mTORC2 activity. This explains the increased phosphorylation of AKT at S473 (an mTORC2-specific phosphorylation site) observed in RHEBL1-knockout MEFs after insulin stimulation . This complex interplay between RHEBL1, mTORC1, and mTORC2 has important implications for diseases involving dysregulated mTOR signaling, including cancer and neurological disorders.

How does RHEBL1 contribute to cancer progression and patient outcomes?

RHEBL1 contributes to cancer progression through multiple mechanisms that influence cellular behavior and patient survival outcomes. In lung cancer cells, RHEBL1 expression is induced by sphingosylphosphorylcholine (SPC), a bioactive lipid elevated in malignant pleural effusions that promotes cancer cell migration .

RHEBL1 overexpression promotes several hallmarks of cancer metastasis:

• Phosphorylation of keratin 8 (K8) at S431 in multiple lung cancer cell lines (A549, H1299, H1703, and H838)

• Reorganization of keratin filaments into perinuclear ring-like structures

• Enhanced cell migration and invasion capabilities even without SPC stimulation

These cellular changes result from RHEBL1's ability to bind and activate AKT1, a central regulator of cell survival and motility. Through this mechanism, RHEBL1 alters the physical properties of cancer cells to promote a more metastatic phenotype.

Clinical data strongly supports RHEBL1's role in cancer progression. Kaplan-Meier survival analysis of lung cancer patients revealed:

These findings suggest that RHEBL1 expression level could serve as a prognostic biomarker in lung cancer. Furthermore, inhibition of RHEBL1 or disruption of RHEBL1-AKT1 binding represents a potential therapeutic strategy for preventing cancer metastasis.

What experimental approaches can effectively measure RHEBL1-AKT1 interaction dynamics?

Investigating RHEBL1-AKT1 interaction dynamics requires a comprehensive experimental toolkit. Co-immunoprecipitation (Co-IP) represents a fundamental approach for detecting protein-protein interactions. Research has successfully employed Co-IP using both AKT1 antibody and RHEBL1 antibody to demonstrate their interaction in cells with and without sphingosylphosphorylcholine (SPC) treatment . This bidirectional Co-IP approach strengthens evidence for genuine interaction.

For visualizing spatial dynamics, confocal microscopy with dual immunofluorescence labeling can reveal co-localization patterns of RHEBL1 and AKT1. This technique confirmed RHEBL1-AKT1 interaction and demonstrated their spatial relationship within cells .

To analyze the structural requirements for interaction, researchers can employ site-directed mutagenesis, generating RHEBL1 mutants with altered G protein activity. Studies using constitutively active (RHEBL1Q64L) and dominant negative (RHEBL1D60K) mutants revealed that G protein activity of RHEBL1 is essential for its binding to AKT1 .

More sophisticated techniques include:

• Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) for real-time monitoring of protein interactions in living cells

• Proximity ligation assay (PLA) to visualize endogenous protein interactions with subcellular resolution

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify binding affinities and kinetics using purified proteins

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

These approaches collectively provide a comprehensive understanding of the spatial, temporal, and structural aspects of RHEBL1-AKT1 interaction dynamics under various physiological and pathological conditions.

How can researchers investigate the role of RHEBL1 in neurological development and myelination?

Investigating RHEBL1's role in neurological development requires specialized approaches focusing on myelination processes. Conditional knockout models using neural progenitor-specific Cre drivers (e.g., Nestin-Cre) have proven effective in studying RHEBL1's neurological functions. The Rheb1f/f, Nestin-Cre mouse model demonstrated that RHEBL1 deletion in neural progenitor cells resulted in abolished mTORC1 signaling and deficits in myelination, despite grossly normal brain development .

Conversely, conditional expression of RHEBL1 transgene in neural progenitors increased mTORC1 activity and promoted myelination, while also rescuing hypomyelination in RHEBL1-knockout mice . This bidirectional manipulation establishes causality in RHEBL1's role in myelination.

For comprehensive analysis of myelination, researchers should employ multiple complementary techniques:

• Biochemical assays: Western blotting to quantify myelin proteins (MBP, PLP, MAG) and phosphorylation of mTORC1 targets (S6, 4E-BP1)

• Histological methods: Immunohistochemistry and electron microscopy to evaluate myelin thickness, compaction, and ultrastructure

• Functional assessments: Electrophysiological recordings to measure conduction velocity in myelinated axons

• Behavioral testing: Motor coordination and cognitive tests to evaluate functional consequences of altered myelination

Cell-specific effects can be investigated using co-culture systems of neurons and oligodendrocytes with cell-specific RHEBL1 manipulation. This approach helps determine whether RHEBL1 functions primarily in neurons, oligodendrocytes, or both during myelination.

For translational relevance, researchers should examine RHEBL1 expression in human samples from patients with demyelinating disorders using immunohistochemistry with RHEBL1 antibodies. These comprehensive approaches will elucidate RHEBL1's role in normal myelination and potential implications for demyelinating disorders.

What are the recommended protocols for studying RHEBL1-mediated effects on keratin phosphorylation and reorganization?

Studying RHEBL1-mediated effects on keratin phosphorylation and reorganization requires a multi-faceted approach. For phosphorylation analysis, Western blotting using phospho-specific antibodies targeting keratin 8 at S431 provides quantitative measurement of keratin phosphorylation status. This technique successfully demonstrated that RHEBL1 overexpression induced K8 phosphorylation in multiple lung cancer cell lines (A549, H1299, H1703, and H838) .

To visualize keratin reorganization, confocal immunofluorescence microscopy is essential. This method revealed that in A549 cells, RHEBL1 localization patterns were similar to keratin filaments, and both reorganized to perinuclear ring-like structures upon sphingosylphosphorylcholine (SPC) treatment . Co-staining for RHEBL1 and keratin enables direct visualization of their spatial relationship during reorganization.

For manipulating RHEBL1 expression, researchers can use:

• Overexpression systems: Transfection with wild-type RHEBL1 or constitutively active (RHEBL1Q64L) constructs

• Gene silencing: siRNA or shRNA targeting RHEBL1

• Pharmacological approaches: Treatment with SPC to induce RHEBL1 expression and keratin reorganization

To elucidate the signaling pathway, inhibitor studies are valuable. Research has shown that MK2206 (an AKT inhibitor) suppressed SPC-induced K8 phosphorylation and reorganization, demonstrating AKT's involvement in this process . Similar approaches can be used with inhibitors of other pathway components to map the complete signaling cascade.

For functional consequences of keratin reorganization, migration and invasion assays should be performed following RHEBL1 manipulation. Transwell migration assays and Matrigel invasion assays have shown that RHEBL1 overexpression promotes these metastasis-associated behaviors .

Live-cell imaging with fluorescently tagged keratin can provide dynamic information about the temporal aspects of reorganization following RHEBL1 manipulation, offering insights beyond static endpoint analyses.

What are the potential therapeutic strategies targeting RHEBL1-AKT1 interaction in cancer?

Targeting the RHEBL1-AKT1 interaction represents a promising therapeutic strategy for cancer treatment, particularly for preventing metastasis. Research indicates that "suppression of RhebL1 or inhibition of RhebL1's binding to AKT1 might be a novel way that prevents changes in the physical properties of metastatic cancer cells" . Several therapeutic approaches warrant investigation:

• Small molecule inhibitors: Developing compounds that specifically disrupt RHEBL1-AKT1 binding could prevent downstream signaling without affecting other RHEBL1 or AKT1 functions. Structure-based drug design targeting the interaction interface would be valuable once the binding domains are fully characterized.

• Peptide-based inhibitors: Designing peptides that mimic critical binding regions could competitively inhibit the RHEBL1-AKT1 interaction. Cell-penetrating peptides could deliver these inhibitory molecules intracellularly.

• Gene therapy approaches: Targeted delivery of RHEBL1 shRNA or siRNA to tumors could reduce RHEBL1 expression. CRISPR-Cas9-based approaches might also allow for selective targeting of RHEBL1 in cancer cells.

• Combination strategies: Since RHEBL1 activates AKT1, combining RHEBL1-targeted therapies with existing AKT inhibitors like MK2206 might produce synergistic effects. Research has shown that MK2206 suppresses SPC-induced effects mediated by RHEBL1-AKT1 signaling .

• G protein activity modulators: Since RHEBL1's G protein activity is crucial for AKT1 binding and activation, developing compounds that selectively inhibit RHEBL1's GTPase activity represents another viable approach.

How might RHEBL1 function in other tissue contexts beyond cancer and neurological development?

RHEBL1's functions likely extend beyond cancer and neurological development to various physiological and pathological contexts. As a regulator of mTORC1 signaling, RHEBL1 likely influences multiple processes including metabolism, immune function, and tissue homeostasis.

In metabolic regulation, RHEBL1 may play roles similar to its paralog Rheb1, which regulates insulin signaling and glucose metabolism. Given that RHEBL1 activates AKT1 , which is central to insulin signaling, RHEBL1 may influence metabolic disorders including diabetes and obesity. Research investigating RHEBL1 expression in metabolic tissues and its response to nutritional status would provide valuable insights.

For immune system function, the mTOR pathway regulates T cell activation, differentiation, and function. RHEBL1's role in regulating mTORC1 suggests potential involvement in immune responses. Studies examining RHEBL1 expression in immune cells and its modulation during immune activation would clarify its immunological functions.

In stem cell biology, mTORC1 signaling influences self-renewal and differentiation. RHEBL1's essential role in embryonic development suggests it may regulate stem cell functions in adult tissues. Investigating RHEBL1 in tissue-specific stem cells could reveal roles in tissue regeneration and homeostasis.

RHEBL1 might also influence aging processes, as mTOR signaling is a well-established regulator of lifespan in multiple organisms. Studies examining age-related changes in RHEBL1 expression and activity could uncover roles in aging-associated diseases.

For cardiovascular function, AKT signaling regulates cardiac hypertrophy and angiogenesis. Given RHEBL1's activation of AKT1 , it may influence cardiovascular pathologies. Examining RHEBL1 expression in cardiac tissues under normal and stress conditions would elucidate potential cardiovascular roles.

These diverse functions highlight the importance of developing tissue-specific conditional knockout models and comprehensive expression analyses to fully characterize RHEBL1's multifaceted physiological roles.