PKC-epsilon regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS

Protein kinase C (PKC) and the actin cytoskeleton are critical effecters of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral me mbrane is poorly defined. In the present study, phorbol 12-myristate 13-ace tate (PMLA) and other activators of PKC selectively enhanced basolateral bu t not apical fluid-phase endocytosis in human T84 intestinal epithelia. Sti mulation of basolateral endocytosis was blocked by the conventional and nov el PKC inhibitor Go-6850, but not the conventional PKC inhibitor Go-6976, a nd correlated with translocation of the novel PKC: isoform PKC-epsilon. PMA treatment induced remodeling of basolateral F-actin. The actin disassemble r cytochalasin D stimulated basolateral endocytosis and enhanced stimulatio n of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane -to-cytosol redistribution of the F-actin cross-linking protein myristoylat ed alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MA RCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A m yristoylated peptide corresponding to the phosphorylation site domain of MA RCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis . MARCKS translocation was inhibited by Go-6850 but not Go-6976. The result s suggest that a novel PKC isoform, likely PKC-epsilon, stimulates basolate ral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.