Figure 3

SUMOylation enhances aPKC activity.

(A) HA-empty vector control, HA-PKCζ-wt, single HA-PKCζ lysine (K) to arginine (R) mutants (K225R, K284R, K378R), double mutants (K225/284R, K284/378R, K378/225R) or triple mutant (K225/284/378R) were co-expressed with FLAG-Lgl1 in HEK293T cells. Cells were lysed, Lgl1 was immunoprecipitated using FLAG-specific antibodies and western analysis of the immunoprecipitates was performed with p-Lgl1/2, HA and FLAG antibodies. The graph represents quantitative data depicting levels of phosphoLgl1 relative to Lgl1 levels. Error bars indicate standard deviations, n = 3, P values were calculated by one-way ANOVA. P value is considered statistically significant if it is less than the critical level and is denoted by *. N.S. indicates non-significant. (B) FLAG IP was performed in lysates prepared from HEK293T cells co-expressing HA-PKCζ-wt or HA-PKCζ-Smut with FLAG-Lgl1. The immunoprecipitates were subjected to WB with HA and FLAG antibodies. The graph represents quantitative data depicting relative interaction of PKCζ with Lgl1. Error bars indicate standard deviations, n = 3, P values calculated by Student’s t test. (C) In vitro kinase assay was performed using in vitro SUMOylated PKCζ. GST-PKCζ was subjected to in vitro SUMOylation reaction using recombinant proteins. The reactions were performed in the presence (+) or absence (−) of ATP (top left panel) and presence (+) or absence (−) of Ubc9 (top right panel). The reactions were subjected to immunoblotting using anti-aPKC antibodies. *indicates SUMOylated PKCζ. The PKCζ from in vitro SUMOylation reactions was further assayed for the activity using a commercial kit as per manufacturer’s instruction. The graph represents quantitative data showing relative activity of PKCζ derived from indicated reactions, expressed as relative luciferase units (RLU). Error bars indicate standard deviations, n = 3, P values calculated by Student’s t test.