Fig. 5.

PKCα and PICK1 cooperatively regulate the Neph1/ZO-1 plasma membrane complex. (A) FLAG-tagged ZO-1^1–503 and V5-tagged Neph1^cyt were coexpressed with PICK1 or empty vector. ZO-1 was precipitated with anti-FLAG antibody. Western blot analysis was performed with a V5-specific antibody to detect coprecipitated Neph protein. Activation of protein kinase C (PKCα) with 200 nM PMA inhibits the interaction (lane 5 and 6). Coexpression of PICK1 further augments the PKC effect on the Neph1/ZO-1 interaction (lane 1 and 2) without affecting protein levels (middle and lower panels). (B) PMA does not influence the PICK1/NEHP1 interaction. Experimental setting as in panel A with the constructs indicated. (C and D) The interaction Neph1/ZO-1 is regulated through PKCα activity. (C) Pulldown assay of bacterially expressed GST.ZO-1^1–111 and MBP.Neph1^cyt in the presence of buffer, PKCα, or calf intestinal alkaline phosphatase (CIAP). Addition of PKCα abrogates the Neph1/ZO-1 interaction, whereas CIAP did not affect the interaction. (D) Kinase assay of bacterially expressed His.Neph1^cyt in the presence of radioactively labeled ATP and buffer, PKCα or CIAP. Addition of PKCα led to phosphorylation of Neph1^cyt. Autoradiograph after in vitro phosphorylation of His.Neph1^cyt (E) Membrane-bound Neph1^cyt (sIg7.Neph1^cyt) or a control plasmid (sIg.7.cdm12) and FLAG-tagged ZO-1^1–503 were expressed in HEK 293T cells and precipitated with protein G. Western blot analysis was performed with a FLAG-specific antibody. Overexpression of PCKα abrograted the Neph1/ZO-1 interaction.