Turnbull, R. E. | Search bioRxiv

bioRxiv2025

NEK7 phosphorylation of cortactin modulates the migratory capacity of cells expressing EML4-ALK V3

EML4-ALK is a common oncogenic driver of non-small cell lung cancer. Distinct EML4-ALK variants cause different rates of disease progression, with patients expressing variant 3 (V3) exhibiting accelerated metastasis. Cells expressing EML4-ALK V3 develop a mesenchymal-like morphology and enhanced migration that is dependent on the NEK9 and NEK7 kinases. However, downstream substrates of these kinases relevant to these phenotypes are largely unknown. Here, we show that the actin-binding protein cortactin is phosphorylated by NEK7 within the F-actin-binding region (ABR) and that depletion of cortactin abrogates the morphological and migration phenotypes induced by EML4-ALK V3. Expression of constitutively active mutants of NEK9 or NEK7 causes similar cortactin-dependent morphological and migration changes. Cortactin co-localises with NEK7 and EML4-ALK V3 at branched filopodia-like extensions that are also generated upon expression of a cortactin protein with phospho-mimetic mutations in the ABR. In contrast, phospho-null mutations dissociate cortactin from F-actin. We propose that EML4-ALK V3 alters cell morphology and promotes directed cell migration by modulating the actin cytoskeleton via NEK7-mediated phosphorylation of cortactin within its ABR. SUMMARY STATEMENTNEK7 phosphorylates key residues of cortactin in its actin-binding repeat region to modulate the actin architecture and migration of cells driven by the EML4-ALK V3 - NEK9 - NEK7 pathway.

cell biology↗

bioRxiv2025

Rapid degradation of 6 class I HDAC complexes reveals minimal functional overlap between complexes

The class 1 HDACs 1, 2 and 3 form seven families of distinct large multiprotein complexes that regulate gene expression via deacetylation of lysines in histone tails. The degree of redundancy and functional overlap between complexes and their primary gene targets, remains unknown. We used CRISPR/Cas9 to independently tag HDAC complexes with FKBP12F36V in HCT116 cells enabling rapid (<1 hr), PROTAC-mediated, degradation. RNA sequencing at 6 h reveals that together, the 4 major complexes (CoREST, NuRD, NCoR/SMRT and SIN3A) perturbed >50% of expressed genes. More than 60% of these are specific to an individual complex. Of genes regulated by more than one complex, approaching 50% are reciprocally regulated such that HDAC complexes act as antagonistic regulators. Homer analysis strongly suggests that the complexes are reliant on different transcription factors. This is the first study to identify the primary targets of individual HDAC complexes and directly compare the effects of rapid degradation on gene regulation in the same biological system.

molecular biology↗