MOBKL1B Human

What is MOBKL1B and what is its biological function?

MOBKL1B, also termed MOB1A, is a mammalian homolog of the yeast Mob and Drosophila MATS (Mob as tumor suppressor) polypeptides. It functions as a component of the protein kinase cascade in the Salvador/Warts/Hippo (SWH) tumor suppressor pathway . MOBKL1B is approximately 25 kDa in size and serves as a preferred substrate for MST1 and MST2 kinases . The protein plays a crucial role in regulating cell proliferation and survival during development. When phosphorylated by MST1/MST2, MOBKL1B alters its binding capabilities to downstream targets such as the NDR-family protein kinases, particularly LATS1 . This interaction is central to the tumor-suppressive function of the Hippo pathway, as MOBKL1B phosphorylation is sufficient to inhibit cell proliferation through actions at several points in the cell cycle.

How is MOBKL1B related to MOBKL1A, and how can researchers distinguish between them?

MOBKL1A and MOBKL1B are highly similar proteins encoded by separate genes in the human genome. MOBKL1A (encoded on Chr 4q13.3) and MOBKL1B (encoded on Chr 2p13.1) exhibit remarkable sequence homology, with 96.3% identity (differing at only 8/216 amino acids) . This high similarity creates significant challenges for researchers attempting to study their individual functions. Standard antibody approaches may not reliably distinguish between these proteins due to their nearly identical epitopes. To properly distinguish between them, researchers should consider:

• Using gene-specific RNA interference with carefully designed siRNAs targeting unique regions

• Employing genomic approaches such as CRISPR-Cas9 to specifically target individual genes

• Creating tagged versions of each protein for overexpression studies

• Using RT-PCR with primers designed to amplify unique regions of each transcript

Since most experimental approaches studying one will inevitably affect or detect the other, researchers should acknowledge this limitation and consider them functionally as MOBKL1A/MOBKL1B in most contexts unless specifically demonstrating isoform-specific effects.

What are the known binding partners of MOBKL1B in signaling pathways?

MOBKL1B interacts with several key proteins as part of its signaling functions:

• MST1 and MST2 kinases - MOBKL1B serves as a substrate for these kinases and undergoes phosphorylation at specific threonine residues (Thr12 and Thr35 in MOBKL1A)

• LATS1 kinase - Phosphorylated MOBKL1B demonstrates enhanced binding to LATS1, which enables LATS1 activation loop phosphorylation in response to stimuli such as H₂O₂

• NDR1 kinase - MST2-catalyzed MOBKL1B phosphorylation enhances binding to NDR1 in vitro

• Viral proteins - Research has explored potential interactions between MOBKL1B and viral proteins such as NS5A from hepatitis C virus, although such findings require careful validation due to potential off-target effects in siRNA experiments

The interaction between MOBKL1B and these binding partners appears to be sequential and regulated by phosphorylation state. For instance, MST2 activation enables its binding to MOBKL1B, and subsequent MST2-catalyzed MOBKL1B phosphorylation inhibits MST2 binding while enhancing MOBKL1B binding to LATS1 .

What are the key phosphorylation sites on MOBKL1B and how do they affect its function?

MOBKL1B undergoes phosphorylation primarily at threonine residues that are critical for its function. Based on studies of MOBKL1A, which is highly homologous to MOBKL1B, the key phosphorylation sites are:

• Threonine 12 (Thr12) - This residue is phosphorylated by MST1/MST2 kinases and contributes to MOBKL1A's ability to bind downstream targets

• Threonine 35 (Thr35) - This is the second major phosphorylation site by MST1/MST2 kinases and appears to have a stronger effect on LATS1 binding compared to Thr12

Functional consequences of these phosphorylation events include:

• Altered binding affinity for MST1/MST2: Phosphorylation reduces the binding of MOBKL1B to MST1/MST2, suggesting a regulatory feedback mechanism

• Enhanced binding to LATS1: Phosphorylation significantly increases MOBKL1B's interaction with LATS1, promoting downstream signaling

• Regulation of cell cycle progression: MOBKL1B phosphorylation impacts both G1/S transition and mitotic exit

The importance of these phosphorylation sites has been demonstrated through mutational studies. When both Thr12 and Thr35 are mutated to alanine (creating a nonphosphorylatable mutant), MOBKL1A/MOBKL1B lose their ability to bind LATS1 effectively, and cells expressing these mutants show accelerated proliferation .

How does MOBKL1B phosphorylation impact cell cycle regulation?

MOBKL1B phosphorylation plays a critical role in regulating multiple phases of the cell cycle:

• G1/S transition: Elimination of MOBKL1B phosphorylation accelerates progression through G1/S into G2/M. This occurs despite relatively low levels of MOBKL1B phosphorylation during G1/S compared to mitosis .

• Mitotic progression: MST1 and MST2 activity increases during mitosis, leading to enhanced MOBKL1B phosphorylation. Both kinases exhibit increased abundance and activation in nocodazole-arrested mitotic cells .

• Mitotic exit: MOBKL1B phosphorylation delays exit from mitosis into G1. Cells expressing nonphosphorylatable MOBKL1B (Thr12/35Ala) show accelerated mitotic exit compared to cells with normal MOBKL1B phosphorylation .

What experimental approaches are most reliable for studying MOBKL1B phosphorylation?

Studying MOBKL1B phosphorylation requires careful experimental design. Based on the literature, the following approaches are recommended:

• Phospho-specific antibodies: Develop antibodies that specifically recognize phosphorylated Thr12 and Thr35 residues of MOBKL1B for western blotting and immunofluorescence studies

• Phosphorylation-specific functional assays:

  • In vitro kinase assays using recombinant MST1/MST2 and MOBKL1B

  • Measuring approximately 1 mol phosphate incorporation per mol of MOBKL1B to assess stoichiometry

  • Binding assays to measure interaction with LATS1 or other partners

• Mutagenesis approaches:

  • Create Thr12Ala, Thr35Ala single mutants and Thr12/35Ala double mutants

  • Analyze these mutants both biochemically and in cellular contexts

  • Note that Thr to Glu mutations do not mimic phosphorylation effects for MOBKL1B

• Cellular stimulation methods:

  • Use nocodazole to arrest cells in mitosis and increase MST1/MST2 activity

  • Apply H₂O₂ or okadaic acid as non-physiological activators of the pathway

  • Monitor both MOBKL1B phosphorylation and subsequent LATS1 activation

• Kinase inhibition/depletion:

  • Use MST2 (K56R) kinase-dead mutants as dominant negatives

  • Apply MST1/MST2-directed shRNAs with at least 90% knockdown efficiency

  • Monitor effects on MOBKL1B phosphorylation in response to stimuli

When designing experiments, researchers should be aware that MST1 and MST2 appear to be the primary or exclusive kinases responsible for MOBKL1B phosphorylation in vivo, as demonstrated by the inhibitory effect of MST2 (K56R) on MOBKL1B phosphorylation in response to diverse stimuli .

What are the considerations for designing siRNA experiments targeting MOBKL1B?

Designing reliable siRNA experiments for MOBKL1B requires careful attention to potential off-target effects, as highlighted by studies in the HCV field . Consider these methodological recommendations:

• Proper control selection:

  • Include seed sequence-matched control siRNAs rather than just irrelevant siRNAs

  • This helps identify off-target effects caused by the seed region of the siRNA

  • Include multiple independent siRNAs targeting different regions of MOBKL1B

• Validation approaches:

  • Confirm knockdown efficiency by both RT-PCR and western blotting

  • Perform rescue experiments using siRNA-resistant MOBKL1B constructs

  • Cross-validate findings using CRISPR-Cas9 or other gene editing approaches

• Specificity considerations:

  • Design siRNAs that can distinguish between MOBKL1A and MOBKL1B despite their high homology

  • Test for effects on the expression of both paralogs

  • Consider the functional redundancy between MOBKL1A and MOBKL1B

• Phenotypic validation:

  • Verify that observed phenotypes correlate with the degree of knockdown

  • Use MOBKL1B mutants (e.g., nonphosphorylatable Thr12/35Ala) to confirm mechanism

  • Examine multiple cellular processes (proliferation, cell cycle, pathway activation)

The importance of these controls is exemplified by a study that initially identified MOBKL1B as important for HCV replication based on siRNA knockdown. Later analysis using seed sequence-matched controls revealed that the observed effect was due to off-target effects of the siRNA rather than specific depletion of MOBKL1B .

How can researchers effectively create and validate MOBKL1B mutants for functional studies?

Creating effective MOBKL1B mutants for functional studies requires strategic design and thorough validation:

• Key mutant designs:

  • Phosphorylation-deficient mutants: Thr12Ala, Thr35Ala, and Thr12/35Ala double mutants

  • Note that phosphomimetic mutations (Thr to Glu) do not effectively mimic phosphorylation of MOBKL1B

  • Binding-interface mutants: Identify and mutate residues involved in LATS1 or MST1/2 binding

• Expression systems:

  • Tetracycline-inducible expression systems to replace endogenous MOBKL1B with mutant versions

  • Combine with shRNAs targeting endogenous MOBKL1A/MOBKL1B for replacement studies

  • Tag mutants appropriately (FLAG, HA, etc.) while ensuring tags don't interfere with function

• Functional validation:

  • Biochemical assays: Assess binding to partners (MST1/2, LATS1) using co-immunoprecipitation

  • Phosphorylation status: Confirm absence of phosphorylation in Ala mutants using phospho-specific antibodies

  • Cellular localization: Verify proper subcellular distribution using immunofluorescence

• Phenotypic analysis:

  • Cell proliferation assays: Measure growth rates over 7-10 days

  • Cell cycle analysis: Use flow cytometry with propidium iodide or FUCCI system

  • Synchronization experiments: Analyze progression through specific cell cycle phases

Researchers have successfully used U2OS cell lines with tetracycline-inducible shRNAs to deplete endogenous MOBKL1A/MOBKL1B while simultaneously expressing recombinant wild-type or mutant MOBKL1A. This approach allowed precise assessment of how MOBKL1B phosphorylation affects cellular processes such as LATS1 activation and cell cycle progression .

How does MOBKL1B integrate into the Hippo pathway signaling cascade?

MOBKL1B serves as a critical adaptor within the Hippo pathway, connecting upstream kinases with downstream effectors:

• Upstream regulation:

  • MST1/MST2 kinases are activated during mitosis or in response to stimuli like H₂O₂ and okadaic acid

  • Activated MST1/MST2 bind to MOBKL1B and phosphorylate it at Thr12 and Thr35

  • This phosphorylation changes MOBKL1B's binding properties, reducing affinity for MST1/MST2

• Downstream signaling:

  • Phosphorylated MOBKL1B exhibits enhanced binding to LATS1 kinase

  • This interaction enables LATS1 activation loop phosphorylation at Ser909

  • LATS1 activation leads to phosphorylation of YAP/TAZ transcriptional co-activators

  • Phosphorylated YAP/TAZ are sequestered in the cytoplasm or degraded, preventing pro-proliferative gene expression

• Pathway dynamics:

  • MOBKL1B phosphorylation appears to be primarily regulated by cell cycle, peaking during mitosis

  • The MST1/MST2-MOBKL1B-LATS1 signaling module regulates both G1/S transition and mitotic exit

  • Disruption of MOBKL1B phosphorylation accelerates cell proliferation by affecting multiple cell cycle phases

This integration positions MOBKL1B as a key mediator that translates MST1/MST2 activation into LATS1 kinase activity, ultimately controlling cell proliferation and organ size through regulation of YAP/TAZ-dependent transcription.

What is the relevance of MOBKL1B in cancer research and potential therapeutic applications?

MOBKL1B's position within the Hippo tumor suppressor pathway makes it highly relevant to cancer research:

• Tumor suppressive mechanisms:

  • MOBKL1B phosphorylation inhibits cell proliferation and is sufficient to delay cell cycle progression

  • Loss of MOBKL1B phosphorylation accelerates proliferation by 2-3 fold, mimicking aspects of cancer cell behavior

  • The MOBKL1B-dependent regulation of LATS1 ultimately controls YAP/TAZ oncogenic activity

• Research considerations for cancer studies:

  • Analyze MOBKL1B phosphorylation status in tumor vs. normal tissue

  • Examine correlations between MOBKL1B function and YAP/TAZ nuclear localization

  • Investigate genetic alterations of MOBKL1B in human cancers

• Therapeutic implications:

  • MOBKL1B represents a potential target for cancer therapy, particularly in cancers with hyperactive YAP/TAZ

  • Strategies to enhance MOBKL1B phosphorylation or mimic its effects on LATS1 could have anti-tumor effects

  • Combination approaches targeting multiple Hippo pathway components may prove most effective

• Methodological approaches:

  • Analysis of MOBKL1B in patient samples using phospho-specific antibodies

  • Correlation of MOBKL1B status with clinical outcomes and other molecular markers

  • In vivo models with tissue-specific manipulation of MOBKL1B phosphorylation status

The research indicates that manipulation of MOBKL1B phosphorylation could potentially alter cancer cell proliferation by affecting both G1/S progression and mitotic exit . This dual-phase regulation makes MOBKL1B an intriguing target for therapeutic development in cancers with Hippo pathway dysregulation.

What are the best methods for detecting MOBKL1B protein and its phosphorylation states?

Researchers studying MOBKL1B should consider these methodological approaches for protein detection:

• Western blotting:

  • Commercial antibodies against MOBKL1B are available from vendors like Cell Signaling Technology

  • Phospho-specific antibodies against Thr12 and Thr35 sites are crucial for studying activation

  • Consider alternative techniques like Phos-tag gels for detecting phosphorylated species

• Immunoprecipitation:

  • Optimize lysis conditions using buffers containing Tris (pH 8.0) and 150mM NaCl

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Consider tandem affinity purification approaches using tagged versions

• Mass spectrometry approaches:

  • Use tryptic digestion followed by ESI-MS-MS to identify phosphopeptides and determine phosphorylation sites

  • Quantitative phosphoproteomics can measure phosphorylation stoichiometry

  • Consider using SILAC or TMT labeling for comparative studies

• Cellular localization:

  • Immunofluorescence using validated antibodies

  • Live-cell imaging with fluorescently tagged MOBKL1B constructs

  • Subcellular fractionation followed by western blotting

• Activity assessment:

  • In vitro binding assays with recombinant LATS1 to assess functional consequences of phosphorylation

  • Cell-based reporter assays measuring YAP/TAZ-dependent transcription as downstream readout

  • Analysis of cell cycle parameters as functional readouts of MOBKL1B activity

When analyzing MOBKL1B phosphorylation, researchers should be aware that MST1/MST2 appear to be the dominant kinases responsible for this modification. The ability of kinase-dead MST2 (K56R) to strongly inhibit MOBKL1B phosphorylation in response to diverse stimuli suggests that other kinases play minimal roles in this process .

How can researchers effectively study MOBKL1B interactions with binding partners?

Studying MOBKL1B protein interactions requires specialized techniques:

• Co-immunoprecipitation approaches:

  • Use antibodies against endogenous proteins or tags on recombinant proteins

  • Compare binding in different conditions (e.g., okadaic acid treatment, mitotic arrest)

  • Analyze how phosphorylation status affects interaction patterns

  • Include appropriate controls (IgG, kinase-dead mutants)

• In vitro binding assays:

  • Express and purify recombinant proteins (MOBKL1B, MST1/2, LATS1)

  • Compare binding of wild-type vs. phosphorylated or mutant MOBKL1B to partners

  • Use GST-pulldown or His-tag pulldown approaches

  • Quantify binding using methods like surface plasmon resonance

• Proximity-based approaches in cells:

  • BioID or TurboID for identifying proximal proteins

  • FRET or BRET to measure direct interactions in live cells

  • PLA (Proximity Ligation Assay) to visualize endogenous protein interactions

• Sequential binding analysis:

  • Design experiments to test the sequential nature of interactions

  • For example, study how MST2-catalyzed MOBKL1B phosphorylation affects subsequent binding to LATS1

  • Use phosphomimetic or phospho-deficient mutants to dissect the sequence of events

• Influence of cellular context:

  • Compare interactions in different cell cycle phases

  • Analyze how stress conditions (H₂O₂, okadaic acid) affect binding patterns

  • Investigate cell type-specific interaction networks

Research has shown that MST2 activation enables its binding to MOBKL1B, and subsequent MST2-catalyzed MOBKL1B phosphorylation inhibits MST2 binding while enhancing MOBKL1B binding to LATS1 . This suggests a sequential binding model where phosphorylation serves as a molecular switch redirecting MOBKL1B from upstream kinases to downstream effectors.

What are the current gaps in MOBKL1B research and emerging areas of investigation?

Despite significant progress in understanding MOBKL1B function, several knowledge gaps and emerging research areas deserve attention:

• Isoform-specific functions:

  • The high homology between MOBKL1A and MOBKL1B (96.3% identity) has complicated the study of their individual roles

  • Future research should develop tools to specifically study each isoform

  • Potential differential regulation or tissue-specific functions remain largely unexplored

• Regulation beyond phosphorylation:

  • While threonine phosphorylation by MST1/MST2 is well-established, other post-translational modifications may exist

  • Transcriptional and epigenetic regulation of MOBKL1B expression remains poorly understood

  • Potential role of non-coding RNAs in regulating MOBKL1B

• Pathway cross-talk:

  • Interaction between MOBKL1B-mediated Hippo signaling and other pathways like Wnt, Notch, or RTK signaling

  • Role of MOBKL1B in coordinating stress responses with cell cycle control

  • Integration of MOBKL1B function with cellular metabolism

• Therapeutic targeting:

  • Development of small molecules that could modulate MOBKL1B function or interactions

  • Strategies to enhance or mimic MOBKL1B phosphorylation for cancer therapy

  • Biomarker potential of MOBKL1B phosphorylation status in cancer diagnosis or prognosis

• Structural biology:

  • Complete structural understanding of MOBKL1B interactions with binding partners

  • Conformational changes induced by phosphorylation

  • Structure-based drug design targeting key interfaces

Future research should aim to develop more specific tools for studying MOBKL1B, including isoform-specific antibodies, CRISPR-based approaches for selective targeting, and improved structural understanding of its interactions. Additionally, exploring MOBKL1B's role in different tissue contexts and disease states beyond cancer would provide a more comprehensive understanding of its biological functions.

How can advanced technologies enhance MOBKL1B research?

Emerging technologies offer new possibilities for understanding MOBKL1B function:

• CRISPR-based approaches:

  • Genome editing to create specific mutations in endogenous MOBKL1B

  • CRISPRi/CRISPRa for modulating expression levels without complete knockout

  • Base editing to introduce specific mutations without double-strand breaks

  • Prime editing for precise modification of phosphorylation sites

• Single-cell technologies:

  • Single-cell RNA-seq to analyze heterogeneity in MOBKL1B expression

  • Single-cell proteomics to measure MOBKL1B protein levels and modifications

  • Live-cell imaging to track MOBKL1B dynamics during cell cycle progression

• Spatial biology techniques:

  • Spatial transcriptomics to map MOBKL1B expression patterns in tissues

  • Multiplexed imaging to analyze MOBKL1B co-localization with binding partners

  • In situ proximity ligation to visualize protein interactions in tissue contexts

• Computational approaches:

  • Machine learning to predict new MOBKL1B interactors or functional relationships

  • Molecular dynamics simulations to understand phosphorylation-induced conformational changes

  • Network analysis to position MOBKL1B within broader signaling networks

• Organoid and in vivo models:

  • Patient-derived organoids to study MOBKL1B function in disease-relevant contexts

  • Conditional knockin/knockout mouse models for tissue-specific manipulation

  • CRISPR screens in vivo to identify genetic interactions with MOBKL1B

These advanced technologies will allow researchers to move beyond reductionist approaches and understand MOBKL1B function in more physiologically relevant contexts. Particularly valuable will be approaches that can distinguish between MOBKL1A and MOBKL1B functions and those that can analyze the dynamic nature of MOBKL1B phosphorylation during normal and pathological cellular processes.