Novel insight into CFTR phosphorylation: implications for its processing, trafficking and function

Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2008Cystic Fibrosis (CF) is the most common lethal monogenic autosomal recessive disease in the Caucasian population and is caused by dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, usually located at the apical membrane of epithelial cells. The most common disease-causing mutation, F508del, causes CFTR protein to be retained at the endoplasmic reticulum (ER) and targeted to proteasomal degradation. Despite great efforts to elucidate the mechanisms and the molecular partners involved in CFTR biogenesis, intracellular localization, trafficking and function, many processes are not fully understood. Protein kinases and phosphatises are known for long to regulate CFTR function (and possibly localization). However, the role of phosphorylation in CFTR biogenesis and trafficking remains uncertain. CFTR processes several CK2 phosphorylation sites (namely one at S511 and another at T1471) and one SYK consensus site at Y512. We produced CFTR mutants in which the consensus residues S511, Y512 and T1471 were substituted by either a neutral (alanine, A) or an acidic residue (aspartic acid, D) in both wt and F508del-CFTR backgrounds and used them to stable transfect BHK cells. Pulsechase experiments followed by CFTR immunoprecipitation and western blot were performed in the procuded cell lines. After quantification of bands B and C of CFTR, results show that whereas substitution of S511 does not affect the turnover or processing of either wt- or F508del-CFTR, mutation of T1471 completely impairs processing of wt-CFTR without affecting significatively F508del-CFTR turnover. However, treatment of cells with 20 uM TBB (tetrabromobenzotriazole , a specific inhibitor of CK2) shows a significant decrease in processing efficiency of wt-CFTR. Furthermore, mutation of putative SYK target Y512 also reduces the steady-state levels of fully processed CFTR without a major impact on F508del-CFTR. Altogether, our data indicate that CK2 and SYK may have a stabilizing role upon wt-CFTR. This effect seems to be independent on residue S511 (or on the putative charge added by aspartic acid replacement at this residue) but apparently dependent on residues Y512 and T1471. However, the effects observed for replacement of these residues suggest that the role of CK2 or SYK is not by direct phosphorylation of CFTR at these positionsResumo alargado disponível em portuguê