CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Microbiomic differences at cancer-prone oral mucosa sites with marijuana usage.

Marijuana smoke contains cannabinoids, immunosuppressants, and a mixture of potentially-mutagenic chemicals. In addition to systemic disease, it is thought to contribute to oral disease, such as tooth loss, tissue changes in the gums and throat, and possibly oral pharyngeal cancer. We used a cross-sectional study of 20 marijuana users and 19 control non-users, to determine if chronic inhalation-based exposure to marijuana was associated with a distinct oral microbiota at the two most common sites of head and neck squamous cell carcinoma (HNSCC), the lateral border of the tongue and the oral pharynx. At the tongue site, genera earlier shown to be enriched on HNSCC mucosa, Capnocytophaga, Fusobacterium, and Porphyromonas, were at low levels in marijuana users, while Rothia, which is found at depressed levels on HNSCC mucosa, was high. At the oral pharynx site, differences in bacteria were distinct, with higher levels of Selenomonas and lower levels of Streptococcus which is what is seen in HNSCC. No evidence was seen for a contribution of marijuana product contaminating bacteria to these differences. This study revealed differences in the surface oral mucosal microbiota with frequent smoking of marijuana.