Specific Translocation of Protein Kinase C to the Plasma Membrane Requires Both Ca²⁺ and PIP₂ Recognition by Its C2 Domain

Supplemental Material

This article contains the following supporting material:

• Figure 2
• Figure 6A
• Figure 6C
• Figure 7A
• Figure 7B
• Figure 7B
• Figure S2
• Figure S3B
• Figure S1 - The PLCδ₁ PH domain and the membrane-targeting domain of GAP43 co-localize at the plasma membrane. Images of cells CFP-PLCδ₁PH (left) and mGAP43-YFP (right) at the apical (top), medial (middle), and basal (bottom) regions of the each cell. The same cell is shown in the middle and bottom panels. Insets show enlarged views of the indicated cell regions. Perinuclear targeting of mGAP43-YFP to Golgi is designated by an asterisk (right images). All results are representative of a minimum of 5 experiments.
• Figure S2 - PKCα C2 domain β strands 3 and 4 are critical for exclusive membrane targeting of hybrid domains. Images of a cell co-expressing YFP-cPLA₂/PKC_CBLs+β3-4 (left) and CFP-cPLA₂/PKC_CBLs (center) at 60 s after stimulation of a cytoplasmic Ca²⁺ signal by treatment with 10 μM ionomycin. A merged image is shown at the right of the panel. Scale bar, 10 μm. Results are representative of a minimum of 5 experiments.
• Figure S3 - The PKCαC2/K209A/K211A and PKCα/K209A/K211A mutants exhibit similar targeting of the TGN. Images of a cell co-expressing (A) YFP-PKCαC2/K209A/K211A (left) and TGN38-CFP (right) or (B) CFP-PKCα/K209A/K211A (left) and YFP-PKCαC2/K209A/K211A (right) 60 and 85 sec, respectively, after stimulation of a cytoplasmic Ca²⁺ signal by treatment with ionomycin. A merged image is shown at the right of each panel. Scale bars, 10 μm. Results are representative of a minimum of 5 experiments.