PKCα phosphorylates DOCK8 for separation from LRCH1. (A) The amino acid sequence (2075–2089) of DOCK8 and its mutants depict the three key serine residues. (B) 293T cells expressing CVIM-FLAG-DOCK8 were untreated or treated with the AKT and PKCα inhibitors, followed by immunoprecipitation with anti-FLAG to detect DOCK8 phosphorylation levels. (C and D) Migration of the T8.1 cells expressing the vector control, DOCK8, or the mutant 3S/E or 3S/A were examined by a transwell assay in response to SDF-1α in the presence or absence of the PKC inhibitor. n = 3. (E) 293T cells were transfected with FLAG-DOCK8 or the 3S/A mutant, followed by a GST-Cdc42G15A pull-down assay to measure their GEF activity. (F) The migration of T8.1 cells coexpressing PKCα with GFP, DOCK8, or the 3S/A mutant was assessed by a transwell assay. n = 3. (G) 293T cells were transfected with HA-LRCH1, FLAG-DOCK8, or 3S/E, followed by immunoprecipitation with anti-FLAG to analyze their binding to LRCH1. (H) The vector control, DOCK8, or the mutant 3S/E were coexpressed with or without LRCH1 into T8.1 cells and migration was examined by a transwell assay in response to SDF-1α. n = 3. (I) The localization of LRCH1 (green), CVIM-DOCK8, or CVIM-DOCK8 3S/A (red) was examined in 293T cells by immunostaining. Bar, 5 µm. (J) 293T cells were transfected with FLAG-DOCK8 or 3S/A together with HA-LRCH1 and Myc-PKCα, stimulated with or without PMA, followed by immunoprecipitation with anti-FLAG to analyze DOCK8 phosphorylation levels and binding to LRCH-1. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. Data are representative of three experiments (C, D, F, H, mean ± SD) or two experiments (B, E, G, and J). Intensity of the immunoblots was quantified and shown at the bottom (mean ± SD). Statistical significance was determined using unpaired Student’s t test.