Differential Gene Expression Analysis Reveals Global LMTK2 Regulatory Network and Its Role in TGF-β1 Signaling

Abstract

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.

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“…Parental HBE CFBE41o-cells, originating from a patient homozygous for F508del mutation (p.F508del/F508del) in the CFTR gene, were maintained in Minimum Eagle's Medium (MEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 50 units/ mL penicillin, 50 μg/mL streptomycin and 2 mM L-glutamine (Thermo Fisher Scientific), and 10% fetal bovine serum (FBS; Corning, NY, USA) in a 5% CO2, 95% air incubator at 37°C [41][42][43]. To establish polarized monolayers, cells were seeded on 24 mm diameter Transwell filters (Corning, New York, NY, USA) at 1 × 10 6 for gene and protein expression or 2 × 10 6 for the small RNAseq experiments and grown in air-liquid interface (ALI) culture at 37°C for 6-9 days.…”

A revised nomenclature for the lemur family of protein kinases

“…The presence and abundance of CFTR targeting LMTK2 in the apical membrane of airway epithelial cells is controlled by Rab11-positive endosomes in a transforming growth factor (TGF)-β1-dependent manner 39 . LMTK2 and TGF-β1 signalling is interwoven in complex signalling cascades as not only TGF-β1 affects LMTK2 signalling but TGF-β1 signalling is also influenced by LMTK2 regulatory network 40 (Fig. 1 ).…”