Protein kinase C-mediated phosphorylation of RKIP regulates inhibition of Na-alanine cotransport by leukotriene D(4) in intestinal epithelial cells.

Leukotriene D4 (LTD4) is an important immune inflammatory mediator that is known to be elevated in the mucosa of chronically inflamed intestine and alter nutrient absorption. LTD4 inhibits Na-alanine cotransport in intestinal epithelial cells by decreasing the affinity of the cotransporter ASCT1. LTD4 is known to increase intracellular Ca(++) and cAMP concentrations. However, the intracellular signaling mechanism of LTD4-mediated ASCT1 inhibition is unknown. In the present study, pretreatment with calcium chelator BAPTA-AM or inhibition of Ca(++)-dependent protein kinase C (PKC), specifically PKCα, resulted in the reversal of LTD4-mediated inhibition of ASCT1, revealing the involvement of the Ca(++)-activated PKC pathway. PKCα is known to phosphorylate Raf kinase inhibitor protein (RKIP), thus activating its downstream signaling pathway. Immunoblotting with anti-RKIP-Ser(153) antibody showed an increase in phosphorylation levels of RKIP in LTD4-treated cells. Downregulation of endogenous RKIP showed no decrease in ASCT1 activity by LTD4, thus confirming its involvement in ASCT1 regulation. Phosphorylation of RKIP by PKC is known to activate different signaling pathways, and in this study it was found to activate cAMP-activated protein kinase A (PKA) pathway. Although protein abundance of ASCT1 was not altered in any of the experimental conditions, there was an increase in the levels of phosphothreonine in ASCT1 protein, thus showing that phosphorylation changes were responsible for the altered affinity of ASCT1 by LTD4. In conclusion, LTD4 inhibits ASCT1 through PKC-mediated phosphorylation of RKIP, leading to the subsequent activation of PKA pathway, possibly through β2-andrenergic receptor activation.