MOBKL3 Human

What is MOBKL3 and what are its alternative nomenclatures in scientific literature?

MOBKL3 (MOB Kinase Activator-Like 3) is also widely known as MOB3, MOB4, PHOCN, PREI3, and CGI-95. It belongs to the Class III/IV subfamily of MOB proteins . The protein is encoded by a gene that produces a 225 amino acid protein with a predicted molecular weight of approximately 25 kDa . MOBKL3 is a component of the cytosolic protein complex and is associated with membranes, particularly at the Golgi apparatus . Unlike some other MOB family members that function as kinase co-activators, MOBKL3 appears to have distinct functions related to membrane trafficking processes, specifically in membrane budding reactions .

How does MOBKL3 differ structurally and functionally from other MOB family proteins?

MOBKL3 belongs to Class III/IV MOB proteins, which differ significantly from Class I and II MOBs in both structure and function. While all MOBs share a conserved Mob/Phocein domain, MOBKL3 and other Class III/IV members lack the capacity for stable binding to NDR kinases, unlike Class I and II MOBs . This fundamental difference directs MOBKL3 toward alternative functions.

Multiple studies have demonstrated that Class III and IV MOBs (including MOBKL3) do not detectably bind to NDR kinases in various independent assays . Instead, MOBKL3 physically associates with other protein complexes such as the STRIPAK complex and appears to function in membrane-associated processes . This distinctive feature places MOBKL3 in a separate functional category compared to the well-characterized Class I MOBs (like MOB1A/B) that serve as critical adaptor proteins in the Hippo signaling pathway .

What protein-protein interactions has MOBKL3 been demonstrated to engage in?

MOBKL3 has been shown to interact with several proteins that suggest its involvement in membrane dynamics and signaling:

• Striatin Family members: MOBKL3 is considered a major partner of Striatin Family proteins, suggesting a role in scaffolding protein complexes .

• DNM1 (Dynamin 1): This interaction implicates MOBKL3 in vesicle formation and membrane fission events .

• EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15): This interaction suggests MOBKL3 may participate in receptor-mediated endocytosis and intracellular trafficking .

• Nucleoside Diphosphate Kinase: This interaction points to potential roles in GTP metabolism which is crucial for membrane trafficking processes .

Unlike the Class I MOB proteins (MOB1A/B) which have well-established interactions with LATS kinases in the Hippo pathway, MOBKL3 appears to function in different molecular contexts, primarily membrane-associated processes .

How do MOB family proteins contribute differently to cellular signaling pathways?

MOB family proteins exhibit remarkable functional diversity within cellular signaling networks:

Class I MOBs (MOB1A/B): Function primarily as adaptors in the canonical Hippo pathway. When phosphorylated by MST1/2, MOB1A/B bind to LATS kinases enabling their autophosphorylation and activation. Active LATS then phosphorylates and inhibits YAP1, the Hippo pathway effector that regulates gene expression . Research has identified numerous interactions for MOB1A/B (48 documented interactions), demonstrating their central role in tissue homeostasis regulation .

Class II MOBs (MOB2): Associate with Tricornered-like NDR family kinases (STK38/STK38L). Multiple studies have confirmed this interaction, though the functional consequences remain somewhat controversial . Some research suggests MOB2 serves as a co-activator, stimulating STK38/STK38L autophosphorylation and increasing kinase activity up to 5.4-fold in experimental settings . The nuclear dbf2-related kinases STK38 and STK38L are consistently identified as top interactors for MOB2 .

Class III/IV MOBs (including MOBKL3): Unlike the other MOB classes, these proteins do not stably bind to NDR kinases . Instead, MOBKL3 appears to function in membrane trafficking processes and has been identified as a component of protein complexes involved in cellular structures and transport mechanisms . This functional distinction highlights how MOB proteins have evolved diverse roles beyond kinase regulation .

What methodologies have proven most effective for studying MOBKL3 protein interactions?

Recent advances in protein interaction methodologies have significantly improved our understanding of MOBKL3. Based on the search results, the following approaches have proven particularly effective:

• Proximity-dependent biotin identification (BioID): This technique has been instrumental in uncovering previously unknown protein interactions of MOB family proteins, including MOBKL3. The approach bypasses limitations of standard protein-protein interaction profiling techniques by labeling proximal proteins in living cells . A comprehensive BioID study of all seven human MOB proteins identified over 200 interactions, with approximately 70% being previously unreported .

• Affinity purification–mass spectrometry (AP-MS) with chemical crosslinking: When investigating interactions within protein complexes, chemical crosslinking prior to immunoprecipitation has proven critical. For example, studies with MOB3C (related to MOBKL3) demonstrated that interactions with the RNase P complex were only detectable when a crosslinking step with dithiobis(succinimidyl propionate) (DSP) was incorporated into the protocol . This suggests that MOBKL3's interactions may similarly require stabilization techniques to be properly detected.

• Co-immunoprecipitation combined with specific tags: Expression systems using tags like 3xFLAG or YFP have enabled detection of specific interactions. Tetracycline-inducible systems (like HeLa Flp-In T-REx cells) provide controlled expression for studying MOBKL3 interactions with minimal background interference .

• Recombinant protein production: E. coli-based expression systems have successfully produced human MOBKL3 recombinant protein with N-terminal His-tags, enabling purification and subsequent interaction studies .

What is the relationship between MOBKL3 and RNA processing complexes?

While direct evidence for MOBKL3's involvement in RNA processing is limited in the provided search results, important insights can be drawn from studies of the closely related MOB3C protein. Recent research has uncovered a surprising connection between MOB family proteins and RNA biology:

MOB3C was discovered to associate with 7 of the 10 protein subunits of the RNase P complex, an endoribonuclease that catalyzes the cleavage of 5' leader sequences from precursor tRNAs . This interaction was confirmed through multiple experimental approaches:

• BioID proximity labeling identified seven RNase P-MRP protein subunits (POP1, POP4, RPP14, RPP25, RPP30, RPP38, and RPP40) as exclusive top candidates in proximity to MOB3C .

• Affinity purification-mass spectrometry with chemical crosslinking validated these interactions .

• Functional analyses confirmed that MOB3C interacts with catalytically active RNase P complex .

Given the sequence similarity between MOB3 subfamily members (72-82% similarity), it is reasonable to hypothesize that MOBKL3 might also interact with RNA processing machinery, though this would require direct experimental validation . The discovery of MOB3C's interaction with RNA processing machinery reveals an unexpected nexus between MOB family proteins and RNA biology that warrants further investigation.

What are the recommended techniques for detecting and analyzing MOBKL3 in tissue samples?

Based on the search results, the following techniques have been validated for MOBKL3 detection:

Immunohistochemistry approaches:

• MOBKL3 polyclonal antibodies are available that specifically detect human MOBKL3 in both standard immunohistochemistry and paraffin-embedded tissue samples . These rabbit polyclonal antibodies have been validated for research applications.

For cellular localization studies:

• Given MOBKL3's association with membranes and Golgi stacks , subcellular fractionation followed by western blotting can be effective for determining its distribution within cellular compartments.

• Immunofluorescence microscopy using validated anti-MOBKL3 antibodies can visualize its native localization patterns, particularly in relation to Golgi markers.

For protein complex analysis:

• Proximity labeling techniques such as BioID have proven particularly valuable for MOB family proteins. This approach involves expressing a bait protein (MOBKL3) fused to a promiscuous biotin ligase, which biotinylates proteins in close proximity .

• For detecting transient or weak interactions, chemical crosslinking with agents like dithiobis(succinimidyl propionate) (DSP) prior to immunoprecipitation significantly improves detection sensitivity .

For quantitative analysis:

• Mass spectrometry-based approaches, particularly when coupled with affinity purification, provide both identification and relative quantification of MOBKL3 and its interaction partners .

How can researchers effectively produce and purify MOBKL3 for biochemical studies?

Recombinant MOBKL3 protein can be effectively produced and purified using the following approach based on available information:

Expression system:

• E. coli has been successfully used as an expression host for human MOBKL3 .

• The expression construct should encode the full human MOBKL3 sequence (Met1-Ala225) with an N-terminal 6His tag to facilitate purification .

Purification protocol:

• Standard His-tag purification protocols using nickel or cobalt affinity chromatography are effective for obtaining highly pure MOBKL3 protein.

• The reported purity of commercially available recombinant MOBKL3 exceeds 95% as determined by SDS-PAGE , suggesting that standard affinity chromatography methods are sufficient.

Quality control:

• SDS-PAGE analysis should be performed to verify protein purity .

• Mass spectrometry can confirm the identity and integrity of the purified protein.

• Functional assays should be developed based on MOBKL3's known binding partners to verify that the recombinant protein retains its native interaction capabilities.

For researchers requiring recombinant MOBKL3 without establishing their own production pipeline, commercially available options include His-tagged human MOBKL3 recombinant protein produced in E. coli .

What experimental approaches can be used to investigate MOBKL3's role in membrane trafficking?

Given MOBKL3's reported associations with membrane trafficking processes , the following experimental approaches would be effective for investigating its functional roles:

Knockdown/knockout studies:

• siRNA or CRISPR/Cas9-mediated depletion of MOBKL3 followed by analysis of membrane trafficking events using fluorescent reporters of endocytosis, exocytosis, or Golgi transport.

• Rescue experiments with wild-type versus mutant MOBKL3 can help identify critical functional domains.

Live-cell imaging approaches:

• Expression of fluorescently-tagged MOBKL3 combined with markers for specific membrane compartments to track its dynamic localization during trafficking events.

• FRAP (Fluorescence Recovery After Photobleaching) analysis to measure MOBKL3 mobility and association with membrane structures.

Interaction studies with trafficking machinery:

• MOBKL3 has documented interactions with DNM1 (Dynamin 1) and EPS15 , which are involved in endocytosis. Co-immunoprecipitation experiments can verify these interactions in different cell types.

• Proximity labeling approaches (BioID/TurboID) with MOBKL3 as bait can identify additional components of trafficking machinery that transiently associate with MOBKL3.

Functional trafficking assays:

• Transferrin or EGF uptake assays to assess endocytosis efficiency in cells with modified MOBKL3 expression.

• VSV-G transport assays to evaluate anterograde trafficking through the Golgi complex.

• FM dye-based assays to measure bulk endocytosis and membrane recycling.

Ultrastructural analysis:

• Electron microscopy of cells with altered MOBKL3 expression to assess morphological changes in membrane compartments, particularly the Golgi apparatus where MOBKL3 has been localized .

How do the functions of human MOBKL3 compare with its homologs in other species?

While the search results don't provide explicit comparative data between human MOBKL3 and its homologs in other species, we can draw some insights about MOB protein evolution and conservation:

The MOB protein family is evolutionarily conserved, with studies in yeast first delineating their functions in kinase pathways regulating cell division and shape . In multicellular eukaryotes, MOB proteins evolved to regulate tissue growth and morphogenesis . This evolutionary conservation suggests that core functions of MOBKL3 may be preserved across species, though species-specific adaptations likely exist.

The classification of MOB proteins into distinct subfamilies (Class I-IV) is consistent across species, indicating functional specialization occurred early in evolution . Human MOBKL3, as a Class III/IV MOB, would likely share functional similarities with its orthologs in other mammals and potentially in more distantly related organisms.

Future comparative studies should focus on:

• Systematic comparison of MOBKL3 interaction networks across model organisms

• Functional conservation of MOBKL3's role in membrane trafficking

• Evolution of MOBKL3's potential involvement in RNA biology processes

What are the most promising future research directions for understanding MOBKL3 function?

Based on the current state of knowledge about MOBKL3 and related MOB proteins, several promising research directions emerge:

Exploring the RNA biology connection:
The discovery that MOB3C (related to MOBKL3) interacts with the RNase P complex opens an exciting new avenue for investigating potential roles of MOBKL3 in RNA processing . Future studies should determine whether MOBKL3 shares this function and investigate the physiological significance of MOB protein involvement in RNA metabolism.

Defining the interactome in different cellular contexts:
Comprehensive proximity labeling studies in diverse cell types could reveal context-specific MOBKL3 interactions, particularly in specialized cells where membrane trafficking is prominent (neurons, secretory cells, etc.) .

Investigating post-translational modifications:
Analysis of how phosphorylation, ubiquitination, or other modifications regulate MOBKL3 function could reveal regulatory mechanisms controlling its activity in membrane trafficking processes.

Therapeutic relevance exploration:
Given the importance of membrane trafficking in diseases ranging from neurodegeneration to cancer, investigating MOBKL3's potential as a therapeutic target or biomarker represents an important translational direction.

Structural biology approaches:
Determining the three-dimensional structure of MOBKL3 alone and in complex with its binding partners would provide crucial insights into its molecular mechanism of action and facilitate structure-based drug design efforts.

What strategies can overcome difficulties in detecting low-abundance MOBKL3 in experimental systems?

Research involving MOBKL3 may face challenges in protein detection due to potentially low endogenous expression levels. The following strategies can help overcome these limitations:

Enhanced immunoprecipitation approaches:

• Chemical crosslinking prior to immunoprecipitation has proven critical for detecting interactions between MOB3C and the RNase P complex . Applying similar crosslinking approaches (using DSP or other crosslinkers) could significantly improve MOBKL3 detection sensitivity.

• Sequential immunoprecipitation protocols may help enrich for low-abundance MOBKL3 complexes.

Signal amplification methods:

• For immunohistochemistry applications, signal amplification techniques such as tyramide signal amplification (TSA) can enhance detection of low-abundance proteins.

• For western blotting, chemiluminescence substrates with extended signal duration may improve sensitivity.

Expression systems for functional studies:

• Tetracycline-inducible systems (such as HeLa Flp-In T-REx cells) allow controlled expression of tagged MOBKL3 for interaction studies while minimizing complications from overexpression .

• Using tags that have been validated with other MOB proteins, such as 3xFLAG or BioID fusions, can facilitate detection while maintaining protein functionality .

Subcellular fractionation:

• Given MOBKL3's association with membranes and Golgi stacks , enriching for these subcellular compartments prior to analysis can concentrate the protein and improve detection sensitivity.

How can researchers reconcile contradictory findings about MOBKL3 function in the literature?

While the search results don't specifically highlight contradictions regarding MOBKL3 function, the broader MOB protein literature contains some contradictory findings, particularly regarding MOB2's role in regulating Tricornered-like kinases . Similar challenges may arise in MOBKL3 research. The following approaches can help researchers address contradictory findings:

Cell type considerations:

• MOB protein functions may vary significantly between cell types. The research should clearly specify the cellular context and avoid generalizing findings across different systems without validation.

• BioID studies of MOB proteins in both HeLa and HEK293 cell lines revealed both common and cell-type specific interactions , highlighting the importance of cellular context.

Experimental conditions:

• MOBKL3 functions may be regulated by cellular conditions like cell cycle stage, stress, or growth factor stimulation. Carefully controlling and reporting these variables helps reconcile apparently contradictory findings.

Protein expression levels:

• Overexpression studies may reveal non-physiological interactions or dominant-negative effects. Comparing results from overexpression systems with those from endogenous protein studies is crucial for accurate interpretation.