The Pseudosubstrate domain of Protein Kinase C-epsilon directs translocation to phagosomes via binding to phosphomonoinositides (135.46)

Protein kinase C-epsilon (PKC-ε) translocation is required for efficient IgG-mediated phagocytosis. We have published that the C1B region, binding to DAG generated by PLCγ1, is necessary for translocation (Cheeseman et al, MCB, 2006). However, C1B is not sufficient for translocation. Deletion and mutational analysis, as well as the use of PKC-ε/PKC-δ chimeras, indicate that the pseudosubstrate domain (εPS) is necessary for PKC-ε translocation. A PKC-ε fragment containing only the C1 and PS regions, as well as a chimera of PKC-δ containing εPS and εC1B, accumulated, demonstrating that these two regions are necessary and sufficient for targeting PKC-ε to phagosomes. Protein-lipid overlays of εPS-GST indicate that εPS preferentially associates with phosphomonoinositides (PI5P, PI3P>PI4P>>>polyphospho-inositides). Of the PI3 Kinase inhibitors, 100 nM wortmannin, but not 50 µM LY294002 inhibits PKC-ε translocation to phagosomes. That both drugs block the formation of PI3P as indicated by 2XFYVE staining argues against PI3P as the docking lipid for PKC-ε. Similar to PKC-ε, wortmannin, but not LY294002, blocked accumulation of PLCδPH (a PI4,5P₂ reporter) at forming phagosomes. Together, these data suggest that εPS binding to PI4P and/or PI5P is necessary for PKC-ε translocation to phagosomes. Efforts are underway to identify the specific lipid binding partner for PKC-ε. Funded by NIH R01 AI05821 (MRL).