Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor.

Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC epsilon was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC epsilon interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC epsilon does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR. In vitro binding assays demonstrate dose-dependent binding of PKC epsilon to RACK1 which is inhibited by an 8-amino acid peptide based on the sequence of the sixth Trp-Asp repeat in RACK1 or by an 8-amino acid sequence in the V1 region of PKC epsilon, epsilon V1-2. A 4-amino acid sequence INAL (70-73) expressed in CFTR shares 50% homology to the RACK1 inhibitory peptide, but it does not bind PKC epsilon. NHERF1 and RACK1 bind in a dose-dependent manner. Immunofluorescence and confocal microscopy of RACK1 and CFTR revealed colocalization of the proteins to the apical and lateral regions of Calu-3 cells. The results indicate the RACK1 binds PKC epsilon and NHERF1, thus serving as a scaffold protein to anchor the enzyme in proximity to CFTR.

Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.