FIGURE 4.

PKC phosphorylation of Ser-922 regulates lipid binding of a C-terminal γ-Pcdh lysine-rich motif. A, PIP Strip (Echelon Biosciences) membranes spotted with phospholipids incubated with the indicated GST fusion proteins and detected with anti-GST antibody. Only proteins with an intact, Ser-922-containing lysine-rich domain exhibited binding to phosphoinositides. PE, phosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PS, phosphatidylserine; Ptdins, phosphatidylinositol; LPA, lysophosphatidic acid; LPC, lysophosphatidyl choline; S1P, sphingosine-1-phosphate. B, micelle binding assay. Shown are absorbance traces from size exclusion chromatography of the indicated GST fusion proteins with or without phosphoinositide micelles. The addition of micelles results in a major shift to larger sizes with intact γC protein but not ΔC11. C and D, co-sedimentation assay. Indicated GST fusion proteins were incubated with crude phosphoinositide micelles before ultracentrifugation to separate the lipids and lipid-associated proteins from the soluble supernatant. Phosphorylation with PKC shifts γC, but not the S/A mutant, from pellet (P) to supernatant (S), indicating reduced phospholipid binding. mAU, milliabsorbance units; WB, Western blot. D shows results from three experiments ± S.E. S/A pellet localization is significantly higher than that of γC. **, p < 0.01.