Figure 5.

PKCι phosphorylates GULP1 T35 and reduces the effect of GULP1 on APP processing. A) Bacterially expressed GST-GULP1_26–43 (WT) or GST-GULP1_26–43 T35A (T35A) were incubated with PKCι immunoprecipitated from transfected cell lysate together with (γ-[³²P])-ATP for 30 min at 30°C. RM is the reaction mix only without kinase. Upper panel: autoradiograph; lower panel: Coomassie Blue staining. PKCι phosphorylates GULP1 at T35. B) HEK293 were cotransfected with APP-GAL4, pFR-Luc, and phRL-TK together with the indicated constructs. PKCι reduces the effect of GULP1-mediated APP-GAL4 cleavage via phosphorylation of T35. ns, not significant (n = 5). Results are means ± sd. **P < 0.01 compared with GULP1 transfected cells. C) Cells were cotransfected with APP and the indicated constructs, and the level of secreted Aβ40 was assayed. PKCι suppresses GULP1-enhanced Aβ liberation through phosphorylation of T35 (n = 5). Results are means ± sd. **P < 0.01 compared with GULP1 transfected cells. D) WT or PKCι knockout (KO) HEK293 cells were cotransfected with APP-GAL4, pFR-Luc, and phRL-TK together with the indicated constructs. GULP1-mediated APP-GAL4 cleavage was enhanced in PKCι KO cells. Left panel: Immunoblot demonstrates KO of PKCι; n = 5. Results are means ± sd. *P < 0.001. E) WT or PKCι KO HEK293 cells were cotransfected with APP and the indicated constructs, and the level of secreted Aβ40 was assayed. GULP1-mediated Aβ40 secretion was increased in PKCι KO cells (n = 5). Results are means ± sd. *P < 0.001. F) PKCι reduces GULP1-APP interaction in coimmunoprecipitation assay. Coimmunoprecipitation was performed from cells transfected with APP + myc-GULP1, APP + myc-GULP1 + PKCι, APP + myc-GULP1 T35A, or APP + myc-GULP1 T35A + PKCι using a mouse anti-myc antibody 9B11. APP and myc-GULP1 in the immunoprecipitates (IPs) were detected by a rabbit anti-APP and a rabbit anti-myc, respectively. Ns, not significant (n = 3). *P < 0.001.