Affinage

NEK4

Serine/threonine-protein kinase Nek4 · UniProt P51957

Length
841 aa
Mass
94.6 kDa
Annotated
2026-04-29
20 papers in source corpus 8 papers cited in narrative 8 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NEK4 is a NIMA-family serine/threonine kinase with threonine phosphorylation preference that operates across multiple cellular systems including ciliogenesis, DNA damage repair, microtubule dynamics, mitochondrial fission, and neuronal circadian signaling (PMID:10529384, PMID:21685204, PMID:22851694, PMID:20103636, PMID:34986513, PMID:39448584). NEK4 localizes to ciliary basal bodies and rootlets through recruitment by RPGRIP1/RPGRIP1L scaffolds, and its depletion impairs cilium assembly (PMID:21685204). It forms a complex with DNA-PKcs, Ku70, and Ku80 to facilitate DNA-PKcs recruitment to double-strand breaks, thereby activating p53 and regulating replicative senescence (PMID:22851694). NEK4 also promotes mitochondrial fission and respiration by phosphorylating DRP1, drives epithelial-to-mesenchymal transition through Smad/Zeb1 signaling, and in neurons alters dendritic spine morphology and circadian phosphoproteomic profiles in a lithium-sensitive manner (PMID:34986513, PMID:30247800, PMID:39448584).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 1999 Medium

    Establishing the enzymatic identity of NEK4: in vitro kinase assays demonstrated that the murine ortholog (STK2) preferentially phosphorylates threonine residues, positioning NEK4 within the NIMA kinase family with a defined substrate specificity.

    Evidence In vitro kinase assay on recombinant murine STK2 with Northern blot expression profiling

    PMID:10529384

    Open questions at the time
    • No physiological substrate identified
    • Threonine preference established only in vitro with artificial substrates
    • No structural basis for substrate selectivity
  2. 2010 Medium

    Revealing a role for NEK4 in microtubule dynamics: a genetic screen showed NEK4 promotes microtubule outgrowth after stabilization and is required for proper G2-M arrest following Taxol treatment, providing the first link between NEK4 and cytoskeletal regulation.

    Evidence shRNA genetic screen, cell cycle analysis, and microtubule outgrowth assays in human cancer cells

    PMID:20103636

    Open questions at the time
    • Direct microtubule-associated substrates of NEK4 not identified
    • Mechanism distinguishing NEK4 effects on stabilized versus destabilized microtubules unknown
    • Single lab study without independent replication
  3. 2011 High

    Placing NEK4 at the cilium: TAP-MS identified NEK4 within RPGRIP1 and RPGRIP1L complexes, and immunofluorescence localized it to basal bodies and ciliary rootlets, while knockdown reduced cilium assembly—establishing NEK4 as a cilium-regulating kinase recruited by RPGRIP scaffolds.

    Evidence Tandem affinity purification/mass spectrometry, siRNA knockdown, and immunofluorescence in ciliated cells

    PMID:21685204

    Open questions at the time
    • Direct ciliary substrates of NEK4 not identified
    • Whether kinase activity is required for ciliogenesis role not tested
    • Relationship between microtubule regulation and cilium function of NEK4 unclear
  4. 2012 High

    Linking NEK4 to DNA damage signaling and senescence: identification of a NEK4–DNA-PKcs–Ku70–Ku80 complex and demonstration that NEK4 depletion impairs DNA-PKcs recruitment to breaks, reduces p53 activation and γH2AX, and extends replicative lifespan.

    Evidence Co-IP/mass spectrometry of NEK4 complexes, γH2AX and p53 phosphorylation assays, DNA-PKcs recruitment assays, and replicative senescence assays in human fibroblasts

    PMID:22851694

    Open questions at the time
    • Whether NEK4 directly phosphorylates DNA-PKcs or Ku subunits not determined
    • Precise step at which NEK4 facilitates DNA-PKcs loading unknown
    • Relative contribution of NEK4 kinase activity versus scaffolding to DNA repair not resolved
  5. 2015 Medium

    Defining isoform-specific interactomes: mass spectrometry of two NEK4 isoforms revealed that the longer isoform (Nek4.1) uniquely associates with RNA splicing factors (SRSF1, SRSF2, SRPK1), implicating a 46-amino-acid insert in expanding NEK4 function to mRNA processing.

    Evidence Co-IP/mass spectrometry comparing Nek4.1 and Nek4.2 interactomes with RNA splicing functional readouts

    PMID:25798074

    Open questions at the time
    • Functional consequences of NEK4–splicing factor interaction on specific transcripts not demonstrated
    • Whether NEK4 phosphorylates splicing factors directly not tested
    • Single lab study
  6. 2018 Medium

    Connecting NEK4 to EMT and metastasis: a kinase-wide siRNA screen identified NEK4 as a positive regulator of EMT, acting through Smad3 and Zeb1 to suppress E-cadherin, with in vivo validation showing reduced metastasis upon NEK4 knockdown.

    Evidence siRNA kinase library screen, gain- and loss-of-function in lung adenocarcinoma cells, western blot for EMT markers, migration/invasion assays, tail-vein metastasis model in mice

    PMID:30247800

    Open questions at the time
    • Direct phosphorylation targets linking NEK4 to Smad/Zeb1 signaling not identified
    • Whether EMT regulation is kinase-activity-dependent not tested
    • Single cancer cell line context
  7. 2022 Medium

    Establishing NEK4 as a regulator of mitochondrial fission and respiration: overexpression enhanced DRP1 phosphorylation and mitochondrial respiration, while a DRP1 inhibitor abolished the respiratory effect, placing NEK4 upstream of DRP1-mediated fission.

    Evidence Overexpression and siRNA knockdown, Seahorse respirometry, DRP1 phospho-western blot, DRP1 inhibitor epistasis experiments

    PMID:34986513

    Open questions at the time
    • Whether NEK4 directly phosphorylates DRP1 or acts through an intermediate kinase (e.g., Erk1/2) not resolved
    • Mitochondrial phenotype not tested in primary cells or in vivo
    • Single lab study
  8. 2024 Medium

    Extending NEK4 function to neuronal circadian biology: forebrain-specific overexpression altered circadian anxiety-like behavior, dendritic spine morphology, and diurnal phosphoproteomic profiles, with lithium rescuing behavioral effects—linking NEK4 to bipolar disorder-relevant circuits.

    Evidence Conditional transgenic and AAV-mediated overexpression in mouse forebrain, behavioral assays, phosphoproteomics, dendritic spine imaging, lithium pharmacological rescue

    PMID:39448584

    Open questions at the time
    • Neuronal substrates of NEK4 responsible for spine and behavioral phenotypes not identified
    • Mechanism of lithium rescue not defined
    • Loss-of-function neuronal phenotype not reported

Open questions

Synthesis pass · forward-looking unresolved questions
  • Despite multiple cellular roles established for NEK4, the direct phosphorylation substrates linking kinase activity to each biological output (ciliogenesis, DNA repair, microtubule dynamics, mitochondrial fission, EMT, neuronal function) remain largely unidentified, and no structural model of NEK4 exists to explain its substrate selectivity or isoform-specific interactions.
  • No direct in vivo substrates identified by phosphosite mapping for any cellular context
  • No crystal or cryo-EM structure of NEK4
  • How a single kinase coordinates such diverse cellular functions (cilium, DNA repair, mitochondria, neurons) mechanistically unresolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005739 mitochondrion 1 GO:0005815 microtubule organizing center 1 GO:0005929 cilium 1
Pathway
R-HSA-1640170 Cell Cycle 1 R-HSA-1852241 Organelle biogenesis and maintenance 1 R-HSA-73894 DNA Repair 1
Partners
DRP1PRKDCRPGRIP1RPGRIP1LSRSF1XRCC5XRCC6YWHAE
Complex memberships
DNA-PK complex (DNA-PKcs/Ku70/Ku80)RPGRIP1/RPGRIP1L ciliary scaffold complex

Evidence

Reading pass · 8 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2011 NEK4 serine/threonine kinase was identified as a component of both the RPGRIP1- and RPGRIP1L-associated protein complexes via tandem affinity purification and mass spectrometry. In ciliated cells, NEK4 localizes to basal bodies, and in ciliated organs it localizes to the ciliary rootlet. Knockdown of NEK4 in ciliated cells significantly decreased cilium assembly, establishing a role for NEK4 in cilium dynamics and suggesting RPGRIP1/RPGRIP1L serve as cilium-specific scaffolds recruiting a NEK4 signaling network. Tandem affinity purification/mass spectrometry, siRNA knockdown, immunofluorescence localization Human molecular genetics High 21685204
2012 NEK4 suppression in human fibroblasts extended replicative lifespan and decreased p21 transcription. Mass spectrometric analysis of NEK4 immune complexes identified a complex containing DNA-PKcs, Ku70, and Ku80. NEK4 suppression impaired recruitment of DNA-PKcs to DNA upon double-strand break induction, resulting in reduced p53 activation and H2AX phosphorylation, establishing NEK4 as a regulator of the DNA damage response and replicative senescence. RNAi screen, Co-IP/mass spectrometry, γH2AX and p53 activation assays, DNA-PKcs recruitment assay Molecular and cellular biology High 22851694
2010 In a genetic screen, NEK4 was identified as a mediator of the response to Taxol (a microtubule stabilizer). After Taxol treatment, NEK4 promoted microtubule outgrowth; NEK4 deficiency impaired G2-M arrest and decreased formation of mitotic-like asters. In contrast, NEK4 deficiency sensitized cells to vincristine (a microtubule destabilizer), demonstrating that NEK4 modulates microtubule dynamics and differentially affects sensitivity to distinct microtubule poisons. Genetic screen (shRNA), cell cycle analysis, microtubule outgrowth assay Cancer research Medium 20103636
1999 Murine STK2 (ortholog of human NEK4) was shown by in vitro kinase assay to phosphorylate substrates on threonine residues (rather than serine), demonstrating substrate specificity similar to NIMA but with a preference for threonine. The protein is expressed ubiquitously and upregulated in testis; its expression was not linked to cell cycle progression in mitogen-activated lymphocytes. In vitro kinase assay, cDNA cloning, Northern blot, immunofluorescence Biochemical and biophysical research communications Medium 10529384
2015 Immunoprecipitation followed by mass spectrometry of two NEK4 isoforms (Nek4.1 and Nek4.2) identified 474 and 149 interacting proteins respectively, including PCNA, DNA-PK components, 14-3-3ε, splicing factors SRSF1, SRSF2, SRPK1, and hnRNPs. The two isoforms showed differing interactomes especially in RNA splicing; Nek4.1 but not Nek4.2 interacted with many splicing proteins, indicating the 46-amino-acid region unique to isoform 1 influences functional context in RNA splicing. Co-IP/mass spectrometry, RNA splicing assays Proteome science Medium 25798074
2018 NEK4 was identified as a positive regulator of epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma cells via a siRNA kinase library screen. NEK4 knockdown increased E-cadherin and ZO1 expression, decreased Zeb1 and Smad proteins, and inhibited cell migration and invasion. NEK4 overexpression decreased E-cadherin and increased Smad3 expression. In vivo tail-vein injection of NEK4-knockdown cells showed reduced tumor formation and metastasis. siRNA kinase library screen, siRNA knockdown, overexpression, western blot, migration/invasion assays, in vivo mouse model Journal of cellular and molecular medicine Medium 30247800
2022 NEK4 overexpression activated mitochondrial respiration coupled to ATP production, increased mitochondrial membrane potential, and enhanced phosphorylation of DRP1 and Erk1/2. NEK4 depletion reduced mitochondrial respiration and impaired mtDNA integrity and caused mitochondrial elongation likely via reduced DRP1 activity. The effects of NEK4 overexpression on mitochondrial respiration were abolished by a DRP1 inhibitor, placing NEK4 upstream of DRP1-mediated mitochondrial fission. Overexpression/siRNA knockdown, Seahorse respirometry, mitochondrial membrane potential assay, DRP1 phosphorylation by western blot, DRP1 inhibitor epistasis The FEBS journal Medium 34986513
2024 NEK4 overexpression in pyramidal neurons of the adult mouse forebrain or dorsal hippocampus (via AAV or transgenic mice) altered circadian fluctuations in anxiety-like behavior, dendritic spine morphology, and synaptic structure. Phosphoproteomic analysis revealed that diurnal and nocturnal phosphoproteomic profiles were different between NEK4 overexpressing and control mice, and lithium treatment ameliorated the behavioral effects of NEK4 overexpression. Conditional transgenic overexpression, AAV overexpression, behavioral assays, phosphoproteomics, dendritic spine imaging Nature communications Medium 39448584

Source papers

Stage 0 corpus · 20 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2011 The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase. Human molecular genetics 58 21685204
2012 Nek4 regulates entry into replicative senescence and the response to DNA damage in human fibroblasts. Molecular and cellular biology 46 22851694
1997 The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae. Molecular and cellular biology 37 9154797
2018 NEK4 kinase regulates EMT to promote lung cancer metastasis. Journal of cellular and molecular medicine 34 30247800
2010 Nek4 status differentially alters sensitivity to distinct microtubule poisons. Cancer research 33 20103636
2015 New interaction partners for Nek4.1 and Nek4.2 isoforms: from the DNA damage response to RNA splicing. Proteome science 25 25798074
1999 NIMA-related kinases: isolation and characterization of murine nek3 and nek4 cDNAs, and chromosomal localization of nek1, nek2 and nek3. Gene 25 10393247
2022 Nek4 regulates mitochondrial respiration and morphology. The FEBS journal 16 34986513
2014 Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2. Biomedical reports 15 25054004
2021 Structure-Activity Relationship (SAR) Study of Spautin-1 to Entail the Discovery of Novel NEK4 Inhibitors. International journal of molecular sciences 8 33435251
2017 Competitive Ability of Maize Pollen Grains Requires Paralogous Serine Threonine Protein Kinases STK1 and STK2. Genetics 8 28986443
1999 Activity and substrate specificity of the murine STK2 Serine/Threonine kinase that is structurally related to the mitotic regulator protein NIMA of Aspergillus nidulans. Biochemical and biophysical research communications 8 10529384
2024 Unraveling NEK4 as a Potential Drug Target in Schizophrenia and Bipolar I Disorder: A Proteomic and Genomic Approach. Schizophrenia bulletin 7 38869147
2022 Anesthetic propofol suppresses growth and metastasis of lung adenocarcinoma in vitro through downregulating circ-MEMO1-miR-485-3p-NEK4 ceRNA axis. Histology and histopathology 6 35521898
2020 Muscle-specific deletion of SLK/Stk2 enhances p38 activity and myogenesis in mdx mice. Biochimica et biophysica acta. Molecular cell research 4 33259860
2024 NEK4 modulates circadian fluctuations of emotional behaviors and synaptogenesis in male mice. Nature communications 3 39448584
2025 NEK4 suppresses cell proliferation in BT20 triple-negative breast cancer cells by diminishing expression of cell cycle genes, while its depletion mitigates proliferation in other cell lines. Frontiers in oncology 1 41001016
2025 NEK4: prediction of available drug targets and common genetic linkages in bipolar disorder and major depressive disorder. Frontiers in psychiatry 0 39950180
2025 A divergent Plasmodium NEK4 acts as a key regulator driving the early events of meiosis. bioRxiv : the preprint server for biology 0 41332573
2025 A divergent Plasmodium NEK4 acts as a key regulator driving the early events of meiosis. Research square 0 41510253