NEK7 Human

What is human NEK7 and what are its primary cellular functions?

Human NEK7 is a regulator of cell division that plays an important role in growth and survival of mammalian cells. It belongs to the NIMA-related kinase family and consists of a conserved C-terminal catalytic domain and a nonconserved, disordered N-terminal regulatory domain . NEK7 functions as a multifunctional kinase, acting in different cellular processes in concert with cell division signaling . Interference with NEK7 function induces growth inhibition, mitotic arrest, and apoptosis, particularly in cervical cancer-derived cell lines like HeLa .

How does the structure of NEK7 contribute to its function?

NEK7 contains two distinct structural regions: (1) a C-terminal catalytic domain that mediates its kinase activity and (2) a nonconserved and disordered N-terminal regulatory domain crucial for protein-protein interactions . The N-terminal domain plays a particularly important role in catalytic regulation, as the truncated version NEK7Δ(1–44) exhibits reduced catalytic activity compared to full-length NEK7 . Both the N- and C-terminal domains are essential structural components for regulation and catalysis, with the disordered N-terminal region enabling binding flexibility with various protein partners .

How does NEK7 differ from its close relative NEK6?

Despite high sequence similarity between NEK6 and NEK7, these kinases display distinct modes of protein interaction and do not share common interactors, with the exception of NEK9 . Experimental evidence using chimeric constructs combining domains from both kinases (N6C7, N7C6) has demonstrated that both the N- and C-terminal domains contribute to their distinct interaction profiles and substrate specificity . The N-terminal domain of NEK7 has a particularly stronger impact on catalytic regulation compared to that of NEK6, with NEK7Δ(1–44) showing markedly reduced activity compared to NEK6Δ(1–33) .

What is known about the NEK7 protein interactome?

The NEK7 interactome has been characterized through complementary approaches: yeast two-hybrid (Y2H) screening and immunoprecipitation followed by mass spectrometry (IP-LC-MS/MS) . These methods have identified 61 NEK7 interactors involved in various biological processes, including cell division . The comprehensive interactome suggests NEK7 functions as a multifunctional kinase in diverse signaling pathways . Key validated interactors include NEK9, RGS2, TUBB, MNAT1, and PLEKHA8, which co-localize with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis .

Which proteins have been validated as NEK7 interactors and potential substrates?

Through additional interaction and phosphorylation assays from yeast two-hybrid screens, CC2D1A, TUBB2B, MNAT1, and NEK9 proteins have been validated as both NEK7 interactors and substrates . The table below shows key NEK7 interactors identified through Y2H screening:

In vitro phosphorylation assays confirmed that NEK7 phosphorylates NEK9, MNAT1, and CC2D1A, but not RGS2 and TUBB2B, indicating selective substrate recognition .

What methodological approaches are recommended for studying NEK7 interactions?

A multi-faceted approach is essential for robust characterization of NEK7 interactions:

• Yeast two-hybrid screening using different tissue-specific cDNA libraries (fetal brain, bone marrow, leukocytes)

• Immunoprecipitation coupled with mass spectrometry (IP-LC-MS/MS) using appropriate controls (e.g., FLAG-control vs. FLAG-NEK7)

• Validation through pull-down assays using 6×His-tagged recombinant proteins with appropriate negative controls

• In vitro phosphorylation assays to distinguish substrate interactions from non-substrate binding partners

• Co-localization studies throughout the cell cycle using immunofluorescence microscopy

• Network analysis of interacting proteins to identify enriched biological processes and cellular components

How should researchers design plasmid constructs for NEK7 expression studies?

For comprehensive characterization of NEK7 functions, researchers should generate multiple constructs:

• Full-length NEK7 can be amplified using primers containing appropriate restriction sites (e.g., BamHI, EcoRI, NdeI, HindIII, SalI) for efficient cloning

• Domain-specific constructs like NEK7Δ(1–44) (deletion of N-terminal regulatory domain) are crucial for understanding domain-specific functions

• Chimeric constructs combining domains from related kinases (e.g., N6C7 containing NEK6 N-terminal domain and NEK7 C-terminal domain) help delineate domain contributions to specificity

• Kinase-dead mutants (e.g., K63M) differentiate between catalytic and scaffolding functions

• Expression vectors should be selected based on the experimental system (bacterial, yeast, mammalian)

What methods are effective for studying NEK7 phosphorylation activity?

To characterize NEK7 kinase activity and substrate specificity:

• In vitro kinase assays using recombinant 6×His-tagged NEK7 and potential substrates

• Standard substrates like ULight-p70S6K peptide (LGFYVAP) can measure relative kinase activity

• Comparison between full-length NEK7 and truncated versions (NEK7Δ(1–44)) reveals the contribution of N-terminal domain to catalytic activity

• Chimeric proteins (N6C7, N7C6) help identify determinants of substrate specificity

• Mass spectrometry can identify specific phosphorylation sites on substrates

• Phospho-null mutants (Ser/Thr to Ala) confirm functional relevance of identified sites

How can researchers differentiate between NEK6 and NEK7 functions despite their high sequence similarity?

Despite their similarity, NEK6 and NEK7 have distinct functions that can be differentiated through:

• Interactome analysis: NEK6 and NEK7 interact with largely different protein sets (NEK9 being a rare exception)

• Domain-swap experiments: Chimeric constructs (N6C7, N7C6) reveal which domains determine specificity

• N-terminal regulatory domain analysis: The N-terminal domains show divergent effects on catalytic activity, with NEK7's having a stronger regulatory effect

• Substrate profiling: In vitro phosphorylation assays with the chimera N6C7 demonstrated ability to phosphorylate both NEK7-specific (NEK9, MNAT1, CC2D1A) and NEK6-specific (SNX26, TRIP4) substrates

• Cellular localization studies: Analysis of subcellular distribution patterns throughout the cell cycle

What role does NEK7 play in mitotic progression?

NEK7 functions as a critical regulator of cell division through several mechanisms:

• NEK7 and its interacting proteins (RGS2, TUBB, MNAT1, NEK9, PLEKHA8) localize at key sites throughout the cell cycle, particularly during mitosis and cytokinesis

• Interference with NEK7 function induces growth inhibition, mitotic arrest, and apoptosis in cell lines like HeLa

• NEK7 phosphorylates substrates involved in cell division signaling, including NEK9 and MNAT1

• The interaction profile of NEK7 shows enrichment for proteins involved in mitotic processes, suggesting coordinated regulation of multiple aspects of cell division

• NEK7's activity appears to be independent of NEK6 in most contexts, indicating non-redundant roles in mitotic progression

How is the NEK9-NEK7 interaction important for mitotic signaling?

The NEK9-NEK7 interaction represents a critical regulatory node in mitotic signaling:

• NEK9's regulatory domain (amino acids 764-976) interacts directly with NEK7

• NEK9 is one of the few proteins that interacts with both NEK7 and NEK6, potentially serving as a common upstream regulator

• NEK9 functions as both an interactor and a substrate of NEK7, suggesting complex regulatory feedback

• Both proteins co-localize during mitosis and cytokinesis, indicating coordinated temporal and spatial regulation

• The interaction was consistently identified across multiple experimental approaches (Y2H, IP-MS, pull-down assays), highlighting its biological significance

• NEK9 was recovered with high redundancy across different cDNA libraries (fetal brain, bone marrow, leukocyte), suggesting conservation of this interaction across tissues

How do the structural domains of NEK7 influence its substrate specificity?

NEK7's substrate specificity is determined by complex interplay between its domains:

• The N-terminal regulatory domain (amino acids 1-44) contributes to substrate recognition despite being disordered

• The C-terminal catalytic domain is essential for phosphorylation activity, but alone is insufficient for proper substrate recognition

• Chimeric constructs (N6C7) can phosphorylate both NEK7-specific substrates (NEK9, MNAT1, CC2D1A) and some NEK6-specific substrates (SNX26, TRIP4), suggesting complex determinants of specificity

• NEK7 interactors often contain disordered regions that may serve as phosphorylation targets

• The full-length NEK7 shows selective substrate phosphorylation, modifying NEK9, MNAT1, and CC2D1A, but not RGS2 and TUBB2B despite interaction with all these proteins

How should researchers interpret domain analysis of NEK7 interacting proteins?

Domain analysis of NEK7 interactors provides crucial insights into interaction mechanisms:

• NEK7 interactors display diverse domain families and disordered segments, suggesting flexibility in recognition mechanisms

• Disordered protein regions enable binding of different partners and may serve as phosphorylation targets

• Analysis of secondary structure and domain composition helps predict potential interaction interfaces

• The variety of domains among NEK7 interactors supports its role as a multifunctional kinase involved in diverse signaling pathways

• Common structural features among interactors may reveal shared recognition motifs for NEK7 binding

What statistical approaches are appropriate for validating NEK7 interactions?

For robust validation of NEK7 interactions:

• In Y2H screens, quantify interaction strength using growth scores on selective media with 3-AT gradient (1-100 mM)

• For IP-MS data, apply stringent filtering to exclude contaminants using resources like the Contaminants Repository for Affinity Purification (CRAPome)

• Prioritize proteins identified in both Y2H and IP-MS approaches as high-confidence interactors

• Validate key interactions through multiple orthogonal techniques (pull-down, co-localization, functional assays)

• Consider redundancy of identification across different tissue libraries as indication of biological relevance

• Apply network analysis to identify functional clusters among interactors

How can researchers distinguish between NEK7 substrates and non-substrate interactors?

To differentiate NEK7 substrates from non-substrate interactors:

• Perform in vitro kinase assays with recombinant proteins (as demonstrated for NEK9, MNAT1, CC2D1A, RGS2, and TUBB2B)

• Identify specific phosphorylation sites using mass spectrometry

• Generate phospho-null mutants to confirm sites and assess functional consequences

• Evaluate whether chimeric constructs (N6C7, N7C6) retain ability to phosphorylate specific substrates

• Consider that interactions may be transient in vivo but detectable by in vitro phosphorylation (as observed with SNX26 and TRIP4)

• Recognize that not all interactors are substrates (e.g., RGS2 interacts with NEK7 but is not phosphorylated by it)

What are common challenges in expressing and purifying active NEK7 protein?

Researchers frequently encounter challenges with NEK7 expression and purification:

• The disordered N-terminal domain may cause protein instability

• Expression levels in bacterial systems may be limited by toxicity

• Maintaining kinase activity throughout purification requires careful buffer optimization

• Different tags and fusion proteins may affect solubility and activity

• Phosphorylation state may influence stability and activity, requiring phosphatase inhibitors during purification

• The NEK7Δ(1–44) construct shows reduced catalytic activity despite improved expression

How can discrepancies between different experimental approaches be reconciled?

To address inconsistencies across experimental platforms:

• Consider technique-specific limitations (e.g., Y2H may detect interactions that don't occur in mammalian cells)

• Validate key findings using multiple orthogonal techniques

• Account for cell-type specificity (interactions may vary between HEK293T and HeLa cells)

• Consider cell cycle dependence of interactions and activities

• Reconcile interaction data with functional outcomes (e.g., interaction without phosphorylation)

• Use chimeric constructs to isolate domain-specific effects when full-length proteins show discrepant results

What controls are essential when studying NEK7 interactions and functions?

Rigorous experimental design requires appropriate controls:

• Include kinase-dead mutants to distinguish catalytic from scaffolding functions

• Use empty vectors/tags (FLAG-control) in immunoprecipitation experiments

• Include negative control proteins (e.g., RARA) and resin-only controls in pull-down assays

• Test Y2H interactions against empty vectors to exclude autoactivation

• Verify specificity by comparing with closely related kinases (NEK6)

• Confirm antibody specificity using knockdown or knockout approaches

• Include chimeric constructs (N6C7, N7C6) to isolate domain-specific contributions