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PCSK9 is implicated in familial hypercholesterolemia via targeting the cell surface PCSK9-LDLR complex toward lysosomal degradation. The M2 repeat in the PCSK9's C-terminal domain is essential for its extracellular function, potentially through its interaction with an unidentified "protein X". The M2 repeat was recently shown to bind an R-x-E motif in MHC-class-I proteins (implicated in the immune system), like HLA-C, and causing their lysosomal degradation. These findings suggested a new role of PCSK9 in the immune system and that HLA-like proteins could be "protein X" candidates. However, the participation of each member of the MHC-I protein family in this process and their regulation of PCSK9's function have yet to be determined. Herein, we compared the implication of MHC-I-like proteins such as HFE (involved in iron homeostasis) and HLA-C on the extracellular function of PCSK9. Our data revealed that the M2 domain regulates the intracellular sorting of the PCSK9-LDLR complex to lysosomes, and that HFE is a new target of PCSK9 that inhibits its activity on the LDLR, whereas HLA-C enhances its function. This work suggests the potential modulation of PCSK9's functions through interactions of HFE and HLA-C. [ABSTRACT FROM AUTHOR]

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called [DELTA]R-Split CFTR, which encodes the front and back halves of CVFR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Co-expression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with [DELTA]R-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of AR-Split/7CA-RD channels was similar to that of [DELTA]R-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions. cystic fibrosis; the cystic fibrosis transmembrane conductance regulator chloride channel; domain-domain interactions; protein kinase A and protein kinase C phosphorylation

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole. Am J Physiol Cell Physiol 279: C108-C119, 2000.--Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [[sup.32]P][PO.sub.4] release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate. cystic fibrosis; phosphorylation; PP2C; (iso)flavonoids; phenylimidazothiazoles; cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). The most common mutation, AF508, causes retention of CFFR in the endoplasmic reticulum (ER). Some CF abnormalities can be explained by altered Ca2+ homeostasis, although it remains unknown how CFTR influences calcium signaling. This study examined the novel hypothesis that store-operated calcium entry (SOCE) through Orail is abnormal in CF. The significance of Orail-mediated SOCE for increased interleukin-8 (IL-8) expression in CF was also investigated. CF and non-CF human airway epithelial cell line and primary cells (obtained at lung transplantation) were used in Ca2+ imaging, electrophysiology, and fluorescence imaging experiments to explore differences in Orail function in CF vs. non-CF cells. Protein expression and localization was assessed by Western blots, cell surface biotinylation, ELISA, and image correlation spectroscopy (ICS). We show here that store-operated Ca2+ entry (SOCE) is elevated in CF human airway epithelial cells (hAECs; ~1.8- and ~2.5-fold for total Ca2+i increase and Ca2+ influx rate, respectively, and ~2-fold increase in the ICRAC current) and is caused by increased exocytotic insertion (~2-fold) of Orail channels into the plasma membrane, which is normalized by rescue of ΔF508-CFTR trafficking to the cell surface. Augmented SOCE in CF cells is a major factor leading to increased IL-8 secretion (~2-fold). CFTR normally down-regulates the Orail/stromal interaction molecule 1 (STIM1) complex, and loss of this inhibition due to the absence of CFTR at the plasma membrane helps to explain the potentiated inflammatory response in CF cells. [ABSTRACT FROM AUTHOR]