MOB1B Human

What is MOB1B and to which protein family does it belong?

MOB1B (Mps One Binder 1B) is a member of the highly conserved, non-catalytic Mob protein family. These proteins were initially discovered as interactors with Mps1 (Monopolar spindle 1) kinase . MOB1B belongs to Class I Mobs, which function as adaptor proteins and co-activators in intracellular signaling pathways, particularly the Hippo pathway. The Mob protein family has expanded in multicellular eukaryotes compared to unicellular yeasts, indicating functional diversification throughout evolution . In humans, MOB1B works in conjunction with its paralog MOB1A, and they often have redundant functions in cellular processes including cell proliferation, cell-lineage specification, and regulation of mitotic exit .

What are the structural features of MOB1B that enable its function?

MOB1B contains a conserved phospho-binding pocket formed by three phosphate-coordinating basic residues (corresponding to Lys-153, Arg-154, and Arg-157 in human MOB1A) . This structural feature enables MOB1B to interact with phosphorylated proteins, particularly STE20 kinases like MST1/2. Critical to its function is the ability to undergo conformational changes upon binding to phosphorylated partners, which facilitates interactions with downstream kinases. The binding interface between MOB1B and NDR/LATS kinases involves specific amino acid contacts, including a hydrogen bond formed between an aspartic acid residue in MOB1B and a histidine residue in LATS kinases (analogous to D63 in MOB1A and H646 in LATS1) . These structural features enable MOB1B to serve as both an allosteric activator and an adaptor protein in multiprotein signaling complexes.

How does MOB1B differ functionally from other MOB family proteins?

MOB family proteins are divided into classes (I-IV) based on sequence divergence and functional specialization. MOB1B, as a Class I Mob, primarily functions as a co-activator of Warts/LATS kinases in the Hippo pathway . Unlike Class II Mobs (like MOB2), which preferentially interact with Tricornered-like kinases (STK38/STK38L), MOB1B makes distinct binding contacts with Warts/LATS kinases. Class III and IV Mobs appear not to bind NDR kinases at all . A striking functional distinction is that the most sequence-divergent class of Mobs are components of the STRIPAK complex, which antagonizes Hippo signaling, contrary to the activating role of MOB1B . These functional differences demonstrate the evolutionary diversification of Mob proteins to regulate various cellular processes through distinct mechanisms.

What is the core function of MOB1B in the Hippo signaling pathway?

MOB1B serves as an essential core component of the Hippo pathway, where it functions as both an adaptor protein and a co-activator of LATS1/2 kinases . The canonical Hippo pathway involves a kinase cascade in which MST1/2 phosphorylates MOB1B, enhancing its binding affinity for LATS1/2 . This interaction leads to full activation of LATS1/2, which in turn phosphorylates the transcriptional co-activators YAP and TAZ, inhibiting their nuclear localization and transcriptional activity . Through this mechanism, MOB1B plays a crucial role in preventing indiscriminate YAP/TAZ hyperactivity, thereby regulating gene expression programs involved in cell proliferation, apoptosis, and differentiation .

How does phosphorylation regulate MOB1B activity in the Hippo pathway?

Phosphorylation of MOB1B by MST1/2 kinases is a critical regulatory mechanism that controls its activity within the Hippo pathway . When MST1/2 is activated, it phosphorylates MOB1B at specific residues, inducing conformational changes that enhance MOB1B's binding affinity for LATS1/2 kinases . Research has demonstrated that this phosphorylation-dependent interaction is required for the full activation of LATS1/2 kinases . The phospho-binding pocket of MOB1B also enables it to recognize and bind phosphorylated residues in the linker domains of upstream kinases, creating a phosphorylation-dependent regulatory network . Methodologically, researchers can study these phosphorylation events using phospho-specific antibodies, phospho-mimetic mutations (e.g., serine/threonine to glutamate), and phospho-dead mutations (e.g., serine/threonine to alanine) to interrogate the functional consequences of MOB1B phosphorylation.

What experimental approaches can be used to manipulate MOB1B activity to study Hippo pathway outputs?

Several experimental approaches can be employed to manipulate MOB1B activity and study its impact on Hippo pathway signaling:

• Genetic manipulation: Conditional knockout or knockdown of MOB1B using CRISPR-Cas9, shRNA, or siRNA techniques can reveal the consequences of MOB1B loss on pathway activity .

• Structure-based mutational analysis: Based on structural insights, researchers can generate specific MOB1B mutants that affect its binding to either upstream kinases (e.g., K104E/K105E mutants that fail to bind Hippo kinases) or downstream effectors (e.g., D63V mutants that affect LATS kinase binding) .

• Phosphorylation site mutants: Creating phospho-mimetic or phospho-dead mutations at key regulatory sites can modulate MOB1B activity.

• Protein-protein interaction modulators: Small molecules or peptides that specifically target MOB1B interactions can be used to disrupt or enhance its function.

• Rescue experiments: As demonstrated in the research with embryonic stem cells, YAP knockdown can rescue defects caused by MOB1A/B depletion, confirming the Hippo pathway dependence of observed phenotypes .

These approaches, individually or in combination, allow researchers to dissect the specific contributions of MOB1B to Hippo pathway signaling and downstream biological processes.

What are the best methodologies for studying MOB1B protein-protein interactions?

Several complementary methodologies are effective for investigating MOB1B protein-protein interactions:

• Co-immunoprecipitation (Co-IP): This technique has been successfully used to detect interactions between MOB1B and its binding partners, including LATS1/2 and MST1/2 kinases . Researchers can use epitope-tagged versions of MOB1B or specific antibodies against endogenous MOB1B.

• Yeast two-hybrid assays: This approach can identify novel interaction partners of MOB1B, similar to how the founding member of the Mob family was discovered as an Mps1 kinase interacting protein .

• Proximity-based labeling: BioID or APEX2-based approaches can identify proteins in close proximity to MOB1B in living cells, potentially revealing transient or context-dependent interactions.

• Förster resonance energy transfer (FRET): By tagging MOB1B and potential binding partners with appropriate fluorophores, researchers can monitor interactions in living cells with high spatial and temporal resolution.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These biophysical methods can determine binding affinities and kinetics of MOB1B interactions with purified proteins.

• Crystallography or cryo-EM: These structural biology approaches can reveal the atomic details of MOB1B-partner complexes, as has been done for MOB1A .

• Competitive binding assays: These have been particularly useful for demonstrating how different Mob proteins can compete for binding to the same partners, as shown for Mob2 outcompeting Mob1A for STK38 binding .

What are the challenges in generating and working with MOB1B knockout models?

Working with MOB1B knockout models presents several technical and biological challenges:

• Functional redundancy: MOB1A and MOB1B have overlapping functions, necessitating double knockout approaches to observe clear phenotypes. Single knockouts may show minimal effects due to compensation .

• Early embryonic lethality: Homozygous deletion of both Mob1a and Mob1b in mice causes lethality at the preimplantation stage, making it difficult to study their function in later development or adult tissues .

• Conditional knockout strategies: To overcome embryonic lethality, conditional knockout systems (like Cre-loxP) must be employed, adding technical complexity to experimental design.

• Tissue-specific effects: MOB1B may have different functions in different tissue contexts, requiring the generation of multiple tissue-specific knockout models.

• Verification of knockout efficiency: Ensuring complete elimination of MOB1B protein, especially in the context of potential compensatory upregulation of MOB1A, requires rigorous validation.

• Distinguishing direct from indirect effects: Because MOB1B affects fundamental cellular processes through the Hippo pathway, distinguishing primary effects from secondary consequences can be challenging.

• Alternative approaches: Researchers have successfully employed conditional depletion in embryonic stem cell models to circumvent some of these challenges, as demonstrated in the study of MOB1A/B's role in stem cell differentiation .

How can researchers quantitatively assess MOB1B's impact on downstream signaling?

Quantitative assessment of MOB1B's impact on downstream signaling can be achieved through multiple approaches:

• Phosphorylation-specific Western blotting: Measuring the phosphorylation levels of downstream targets like YAP (at serine 127) and TAZ using phospho-specific antibodies can provide a direct readout of pathway activity .

• Nuclear/cytoplasmic fractionation: Quantifying the distribution of YAP/TAZ between nuclear and cytoplasmic compartments can assess the functional outcome of MOB1B-mediated regulation.

• Luciferase reporter assays: TEAD-responsive luciferase reporters can measure YAP/TAZ-dependent transcriptional activity as a functional readout of MOB1B's impact on Hippo signaling.

• RT-qPCR of target genes: Quantifying the expression of YAP/TAZ target genes (such as CTGF, CYR61, and ANKRD1) provides a functional measure of pathway output.

• Phospho-kinase activity assays: In vitro kinase assays using immunoprecipitated LATS1/2 can directly measure how MOB1B affects kinase activity.

• Phosphoproteomics: Mass spectrometry-based phosphoproteomics can provide a global view of phosphorylation changes downstream of MOB1B manipulation.

• Live-cell imaging: Using fluorescent reporters of YAP/TAZ localization or activity can provide dynamic, single-cell measurements of MOB1B's impact on signaling.

These quantitative approaches, particularly when used in combination, enable researchers to comprehensively assess how MOB1B influences Hippo pathway signaling.

How does MOB1B contribute to embryonic stem cell differentiation?

Research has demonstrated that MOB1B, together with MOB1A, plays a critical role in the differentiation of embryonic stem cells (ESCs) into the three germ layers . While Mob1a/b depletion does not affect the stemness or proliferation of mouse ESCs, it causes significant defects in their ability to differentiate. This regulation operates through the Hippo pathway, as evidenced by the finding that Yap knockdown can rescue the differentiation defects caused by Mob1a/b depletion . This suggests that MOB1A/B normally restrains YAP activity to permit proper differentiation. The mechanism likely involves MOB1B's role in activating LATS1/2 kinases, which phosphorylate and inhibit YAP/TAZ, allowing the expression of differentiation-associated genes. Methodologically, researchers used conditional depletion systems to study MOB1A/B function in ESCs, coupled with in vitro differentiation assays and in vivo teratoma formation experiments to assess differentiation capacity across all three germ layers .

What experimental systems best model MOB1B function during early embryonic development?

Several experimental systems can effectively model MOB1B function during early embryonic development:

• Conditional knockout embryonic stem cells: As demonstrated in the research, ESCs with conditional depletion of Mob1a/b provide a versatile system to study MOB1B's role in pluripotency and differentiation .

• Teratoma formation assays: Injecting ESCs into immunodeficient mice to form teratomas allows assessment of their differentiation potential into all three germ layers in vivo .

• Embryoid body formation: This in vitro differentiation system recapitulates aspects of embryonic development and can reveal stage-specific requirements for MOB1B.

• CRISPR-Cas9 genome editing in zygotes: Despite early lethality challenges, precise timing of MOB1B disruption can provide insights into its earliest developmental functions.

• Chimeric embryo analysis: Combining MOB1B-deficient cells with wild-type cells in chimeric embryos can reveal cell-autonomous versus non-autonomous functions.

• Conditional knockout mouse models: Tissue-specific or temporally controlled deletion of MOB1B can circumvent early embryonic lethality.

• Species-specific models: Comparing MOB1B function across species (mouse, zebrafish, Xenopus) can reveal evolutionarily conserved and divergent roles.

These systems, particularly when used in combination, provide complementary insights into MOB1B's developmental functions.

What is the relationship between MOB1B activity and lineage-specific differentiation programs?

The relationship between MOB1B activity and lineage-specific differentiation programs is complex and context-dependent:

• Germ layer specification: MOB1B depletion causes defects in differentiation into all three germ layers (ectoderm, mesoderm, and endoderm), suggesting a fundamental role in early lineage commitment .

• YAP/TAZ-dependent mechanisms: MOB1B regulates differentiation primarily through modulation of YAP/TAZ activity. Hyperactivation of these factors due to MOB1B loss inhibits proper differentiation programs .

• Interaction with developmental signaling pathways: MOB1B may influence lineage specification by modulating cross-talk between the Hippo pathway and other developmental signaling networks (e.g., TGFβ, Wnt, Notch).

• Temporal dynamics: The requirement for MOB1B activity likely varies during different stages of differentiation, with critical windows for its function.

• Tissue-specific roles: While MOB1B affects all three germ layers, the molecular mechanisms and target genes influenced may differ between lineages.

• Epigenetic regulation: MOB1B-mediated regulation of YAP/TAZ may influence chromatin accessibility and epigenetic modifications that control lineage-specific gene expression programs.

Future research employing lineage-specific reporters, single-cell transcriptomics, and chromatin profiling will further elucidate the precise mechanisms by which MOB1B influences different differentiation programs.

How are MOB1B mutations or dysregulation linked to human cancers?

Altered regulation of or mutations in human MOB genes, including MOB1B, are associated with numerous cancers . As a key component of the tumor-suppressive Hippo pathway, MOB1B dysregulation can lead to aberrant cell proliferation and tumorigenesis through several mechanisms:

• Direct gene mutations: Mutations that impair MOB1B's ability to activate LATS1/2 kinases can lead to unrestrained YAP/TAZ activity and oncogenic transformation.

• Expression alterations: Downregulation of MOB1B expression through epigenetic mechanisms or post-transcriptional regulation can diminish Hippo pathway tumor-suppressive functions.

• Post-translational modifications: Aberrant phosphorylation or other modifications of MOB1B can affect its activity and interaction with binding partners.

• Pathway cross-talk: Disruption of MOB1B-mediated Hippo signaling can affect its integration with other cancer-relevant pathways, including Wnt, TGFβ, and Ras signaling.

Methodologically, cancer researchers investigate MOB1B's role through genetic screening of human tumors, functional studies in cancer cell lines, patient-derived xenografts, and genetically engineered mouse models. Since MOB1B and MOB1A have overlapping functions, studies often need to address both proteins to fully understand their tumor-suppressive roles.

What are the methodological approaches for targeting MOB1B therapeutically in cancer?

Several methodological approaches could be employed to target MOB1B therapeutically in cancer contexts:

• Small molecule modulators: Developing compounds that enhance MOB1B's ability to activate LATS1/2 kinases could restore tumor-suppressive Hippo signaling in cancers with pathway dysregulation.

• Protein-protein interaction stabilizers: Molecules that stabilize the interaction between MOB1B and LATS1/2 could enhance pathway activity.

• Gene therapy approaches: Restoration of MOB1B expression in tumors with decreased levels could reactivate Hippo signaling.

• Synthetic lethality strategies: Identifying genes that, when inhibited, cause selective death of cells with MOB1B dysregulation.

• Combination therapies: Targeting MOB1B in conjunction with other Hippo pathway components or parallel oncogenic pathways may enhance therapeutic efficacy.

• Peptide-based approaches: Developing peptides that mimic MOB1B functional domains to compete with endogenous disrupted interactions.

• RNA-based therapeutics: Using antisense oligonucleotides or siRNAs to modulate MOB1B expression or splicing in context-specific manners.

The development of these approaches requires detailed structural understanding of MOB1B interactions, validation in preclinical models, and consideration of potential compensatory mechanisms involving MOB1A.

How can researchers distinguish between the roles of MOB1A and MOB1B in tumor suppression?

Distinguishing between the roles of MOB1A and MOB1B in tumor suppression presents a significant challenge due to their functional redundancy but can be approached through several methodological strategies:

• Individual versus combined knockouts: Comparing phenotypes of MOB1A-only, MOB1B-only, and double knockout models can reveal unique and shared functions .

• Expression pattern analysis: Examining differential expression patterns of MOB1A and MOB1B across tissues and tumor types may indicate context-specific roles.

• Paralog-specific mutations: Introducing mutations that specifically affect one paralog but not the other can help delineate their unique functions.

• Rescue experiments: Testing whether MOB1A can rescue MOB1B deficiency (and vice versa) in different contexts can reveal functional equivalence or specificity.

• Interaction proteomics: Identifying unique binding partners for each paralog may reveal divergent signaling roles.

• Structure-function studies: Detailed analysis of structural differences between MOB1A and MOB1B can highlight potential functional distinctions.

• Patient data correlation: Analyzing correlations between MOB1A versus MOB1B alterations and clinical outcomes in cancer patients may reveal paralog-specific prognostic significance.

These approaches, especially when combined, can help researchers decipher the potentially distinct contributions of MOB1A and MOB1B to tumor suppression across different cancer contexts.

How might post-translational modifications beyond phosphorylation regulate MOB1B function?

While phosphorylation is the best-characterized post-translational modification (PTM) of MOB1B, other modifications likely play important roles in regulating its function:

• Ubiquitination: This modification could regulate MOB1B protein stability, subcellular localization, or interaction capabilities. Researchers should investigate whether E3 ubiquitin ligases target MOB1B and under what conditions.

• SUMOylation: This modification often affects protein-protein interactions and subcellular localization, potentially influencing MOB1B's ability to form functional complexes.

• Acetylation: Lysine acetylation could alter MOB1B's binding properties or stability, particularly given the importance of lysine residues in its phospho-binding pocket .

• Methylation: This modification might fine-tune MOB1B's interaction with binding partners or influence its subcellular distribution.

• O-GlcNAcylation: As a nutrient-responsive modification, this could connect MOB1B function to cellular metabolic state.

Methodologically, mass spectrometry-based proteomics, site-directed mutagenesis, and modification-specific antibodies can be employed to identify and characterize these PTMs. Researchers should particularly focus on how these modifications might be altered in disease states or in response to cellular stressors.

What is the significance of MOB1B in non-canonical Hippo pathway signaling?

MOB1B likely plays important roles in non-canonical Hippo pathway signaling that extend beyond the classical MST1/2-LATS1/2-YAP/TAZ cascade:

• Interaction with alternative kinases: Research should investigate whether MOB1B interacts with and regulates MAP4K- and GckIII-group GCK kinases, which can activate LATS1/2 independently of MST1/2 .

• STRIPAK complex regulation: Since some Mob family members are components of the STRIPAK complex that antagonizes Hippo signaling, potential cross-regulation between MOB1B and this complex merits investigation .

• Cross-talk with other signaling pathways: MOB1B may serve as a node for integration of Hippo signaling with other pathways such as Wnt, Notch, and TGFβ signaling.

• Cytoskeletal regulation: Given the role of Hippo signaling in mechanotransduction, MOB1B may link mechanical cues to cellular responses through non-canonical mechanisms.

• Mitotic functions: Based on the original identification of Mob proteins as regulators of mitosis, MOB1B might have YAP/TAZ-independent functions in cell division .

Advanced methodologies including proximity labeling proteomics, pathway-specific reporters, and systematic genetic interaction screens could help uncover these non-canonical functions.

How does the tissue microenvironment influence MOB1B functionality in vivo?

The tissue microenvironment likely exerts significant influence on MOB1B functionality through multiple mechanisms:

• Mechanical forces: As the Hippo pathway responds to mechanical cues, physical properties of the microenvironment (stiffness, topology) may modulate MOB1B-dependent signaling.

• Cell-cell contacts: The density and type of cell-cell interactions could affect MOB1B activation state and localization, particularly in epithelial tissues.

• Soluble factors: Growth factors, cytokines, and hormones present in the microenvironment may influence MOB1B through effects on upstream regulators.

• Nutrient availability: Metabolic state of the tissue could affect MOB1B function through energy-sensing pathways or direct post-translational modifications.

• Extracellular matrix composition: Different ECM components might trigger specific signaling cascades that converge on MOB1B regulation.

• Tissue-specific interaction partners: MOB1B may engage with different proteins depending on the cell type and tissue context.

Investigating these questions requires advanced in vivo models with tissue-specific manipulation of MOB1B, intravital imaging techniques to visualize signaling dynamics, and organoid systems that recapitulate tissue architecture. Single-cell approaches would be particularly valuable for capturing the heterogeneity of MOB1B function within complex tissue environments.

What are the most promising translational applications of MOB1B research?

The research on MOB1B has several promising translational applications:

• Regenerative medicine: Understanding MOB1B's role in stem cell differentiation could inform strategies for directed differentiation of stem cells for tissue engineering and cell therapy applications .

• Cancer therapeutics: As a tumor suppressor component of the Hippo pathway, targeting MOB1B or its downstream effectors could yield new approaches for cancer treatment, particularly in tumors with dysregulated YAP/TAZ activity .

• Developmental disorders: Insights into MOB1B's function in embryonic development could shed light on congenital disorders resulting from disrupted differentiation programs .

• Tissue repair and fibrosis: Modulating MOB1B activity might help regulate YAP/TAZ in contexts of wound healing and fibrotic disease, where these factors play key roles.

• Diagnostic biomarkers: Patterns of MOB1B expression or post-translational modification could serve as biomarkers for disease states or treatment response.

Advancing these translational applications will require continued basic research into MOB1B's molecular mechanisms alongside targeted preclinical studies in disease-relevant models.

What technological advances would most accelerate research into MOB1B function?

Several technological advances would significantly accelerate research into MOB1B function:

• Structural biology tools: Improved techniques for solving structures of MOB1B in complex with its various binding partners would provide insights for rational drug design and mechanistic understanding.

• Optogenetic and chemogenetic tools: Development of tools to spatiotemporally control MOB1B activity would allow precise dissection of its functions in different cellular compartments and developmental stages.

• Single-molecule imaging: Technologies that enable visualization of MOB1B interactions and conformational changes in living cells would reveal dynamic aspects of its function.

• Protein engineering approaches: Creating MOB1B variants with altered specificity or novel functions could help dissect its various roles and potentially lead to therapeutic applications.

• AI-driven predictive modeling: Computational approaches to predict MOB1B's interaction network and functional outcomes of its perturbation could guide experimental design.

• Genome-wide CRISPR screens: Systematic identification of genes that modify MOB1B-dependent phenotypes would reveal new pathway components and regulatory mechanisms.

• Patient-derived models: Development of improved models derived from patient cells or tissues would enhance translational relevance of MOB1B research.