Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum

CFTR possesses a large cluster of strict dibasic consensus sites for phosph orylation by protein kinase A (PKA) in the R-domain and an obligatory depen dence on phosphorylation is a hallmark of CFTR Cl- channel function. Remova l of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been repo rted, leaving open the possibility that the residual response may be mediat ed by associating ancillary phosphoproteins. We traced the residual PKA-cat alyzed P-32-labelling of the variant with 11 sites mutagenized (11SA) to di stinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additiona l monobasic sites in these segments produced a 15SA variant in which Cl- ch annel response to PKA was abolished. Therefore, it can be concluded that an cillary phosphoproteins do not contribute to CFTR activation by PKA. Notabl y, however, the 15SA protein did exhibit a low level of constitutive channe l activity not dependent on PKA, which might have reflected a down-regulati ng effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case. Since immature CFTR has been claimed to be active in the endoplasmic reticu lum (ER), we also examined whether it can be phosphorylated in cells and wh at influence if any this might have on its susceptibility to degradation. T eleologically, activation by phosphorylation of CFTR Cl- channels in the ER might be undesirable to the cell. Using various phosphorylation site mutan ts and kinase and phosphatase inhibitors in pulse-chase experiments, we hav e found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore , we found that microsomes from cells expressing CFTR processing mutants su ch as Delta F508 do not generate Cl- active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascen t chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containi ng vesicles into the plasma membrane as part of the mechanism of stimulatio n of chloride secretion has been reported, as has an influence of CFTR on t he balance between endocytosis and exocytosis but these findings have not b een universally confirmed. (C) 1999 Elsevier Science B.V. All rights reserv ed.