Journal: The Journal of Biological Chemistry

Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells *

doi: 10.1074/jbc.M114.563742

Figure Lengend Snippet: Experiments demonstrating that LMTK2 localizes at the plasma membrane and co-immunoprecipitates with CFTR in human airway epithelial cells. A , schematic illustration of domain organization in CFTR and LMTK2. CFTR: TMD , transmembrane domain; NBD , nucleotide binding domain; R , regulatory domain. The R contains the phosphor-specific inhibitory Ser 737 site and other PKA consensus sites. LMTK2: TMD , transmembrane domain; KD , kinase domain. The residue Lys 168 located upstream of the Walker A motif is critical for kinase activity ( , ). MBD , myosin binding domain. Amino acid residues 567–773 mediate direct binding to myosin VI . TD , tail domain. B and C , immunoblots demonstrating similar distribution of LMTK2 at the plasma membrane of primary differentiated HBE cells and polarized human bronchial epithelial cell model CFBE41o- cells. The apical ( AP ) or basolateral ( BL ) plasma membrane ( PM ) proteins were isolated by domain-selective cell surface biotinylation in monolayers cultured on separate Transwell permeable growth supports. The whole cell lysate ( WCL ) and PM fraction of apically or basolaterally biotinylated monolayer were labeled AP and BL , respectively. Epithelial cell polarization is demonstrated by the basolateral localization of Na,K-ATPase. LMTK2 was detected at both membrane domains with AP:BL ratio of ∼1:1 when normalized for the corresponding LMTK2 abundance in WCL. WCL represents 5% of BT sample. D and E , immunoprecipitation experiments demonstrating that LMTK2 and CFTR co-immunoprecipitate in Calu-3 cells. CFTR was immunoprecipitated with the mouse monoclonal antibody M3A7 (IP CFTR, D ), and LMTK2 was immunoprecipitated with the rabbit polyclonal anti-LMTK2 antibody (IP LMTK2, E ). Mouse or rabbit non-immune IgGs were used as controls (IP IgG). WCL represents 2% of IP sample. Proteins were separated by SDS-PAGE using 7.5% gels and analyzed by immunobloting ( IB ) as indicated. All experiments were repeated three times from separate cultures with similar results.

Article Snippet: The following anti-human CFTR antibodies were used: mouse monoclonal, clone 596 (Cystic Fibrosis Foundation Therapeutics, Inc.; Chapel Hill, NC) and clone M3A7 (Millipore; Billerica, MA), and rabbit polyclonal Ab-737 (Assay Biotechnology Inc. San Francisco, CA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Residue, Activity Assay, Western Blot, Isolation, Cell Culture, Labeling, Immunoprecipitation, SDS Page