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Lemur tyrosine kinase 2 (LMTK2) is a transmembrane Ser/Thr kinase whose role has been increasingly recognized; however, when compared to other kinases, understanding of the LMTK2 networks and biological functions is still limited. Recent data have shown that transforming growth factor (TGF)-β1 plays a role in modulating LMTK2 function by controlling its endocytic trafficking in human bronchial epithelial cells. Here, we aimed to unveil the LMTK2 regulatory network and elucidate how it affects cellular functions and disease pathways in either TGF-β1 dependent or independent manner. To understand how the LMTK2 and TGF-β1 pathways interconnect, we knocked down (KD) LMTK2 using small(si)RNA-mediated silencing in human bronchial epithelial CFBE41o- cells, treated cells with TGF-β1 or vehicle control, and performed differential gene expression analysis by RNA sequencing (RNAseq). In vehicle-treated cells, LMTK2 KD affected expression of 2,506 genes while it affected 4,162 genes after TGF-β1 stimulation. Bioinformatics analysis shows that LMTK2 is involved in diverse cellular functions and disease pathways, such as cell death and survival, cellular development, and cancer susceptibility. In summary, our study increases current knowledge about the LMTK2 network and its intersection with the TGF-β1 signaling pathway. These findings will serve as basis for future exploration of the predicted LMTK2 interactions and signaling pathways.

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl– channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-β1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl– transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-β1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-β1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.