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Figure 5


Fig. 5. The binding of 14-3-3 proteins to PI4KIIIß stabilizes Ser294 phosphorylation. (A) Flag-tagged wildtype PI4KIIIß expression vector was transiently transfected into HEK293 cells along with vector, HA-tagged 14-3-3{tau} or HA-tagged DN-14-3-3{tau}, respectively. Flag-PI4KIIIß was precipitated using Flag-specific antibodies and precipitates were immunoblotted with pMOTIF, Flag and HA-specific antibodies. To verify HA-14-3-3{tau} expression total cell lysates (TCL) were immunoblotted with HA-specific antibodies. (B) HEK293 cells were transfected with plasmids encoding the indicated proteins, lysed and PKD1-GFP was precipitated using anti-GFP antibodies. Precipitates were subjected to in vitro kinase assay as described in Materials and Methods. (C) HEK293 cells were transfected with the Flag-PI4KIIIß expression construct and Flag-PI4KIIIß was purified with Flag-M2-Agarose. The purified enzyme was either incubated with PBS, 4 µg of GST, 4 µg of GST-DN-14-3-3{tau} or 1 µg and 4 µg of GST-14-3-3{tau} on ice overnight. The mixture was then incubated with or without 10 units of {lambda} phosphatase, and dephosphorylation was performed at 30°C for 30 minutes. The phosphorylation status of Ser294 in Flag-PI4KIIIß was detected by immunoblotting with anti-PKD pMOTIF antibody; blot was further detected for Flag-PI4KIIIß. GST fusion proteins were visualized by Ponceau-S staining.