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First published online 19 November 2003
doi: 10.1242/jcs.00832

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Targeting of PKC and in the pituitary: a highly regulated mechanism involving a GD(E)E motif of the V3 region

Corinne Quittau-Prévostel, Nathalie Delaunay, Alejandra Collazos, Alice Vallentin^* and Dominique Joubert

Inserm U469 `Molecular and Cellular Endocrinology: Signaling and Pathology', 141 rue de la Cardonille, 34094 Montpellier CEDEX 05, France

View larger version (27K): [in a new window]   Fig. 1. Schematic view and expected molecular size (A,B) of the generated GFP-tagged wild-type and mutated PKC. (C,D) Western blot analysis of the fusion proteins transiently transfected in the GH3B6 cell line. A positive immunoreactive band was evidenced for each GFP tagged protein at the appropriate molecular size.

View larger version (66K): [in a new window]   Fig. 2. PKC and laminin immunostaining of sagittal sections (40 µm) of unstimulated and TRH-stimulated rat pituitary. Pituitary glands were removed from the animal and directly incubated in HamF10 culture medium containing 100 nM TRH for 1 hour. Pituitary glands were fixed in paraformaldehyde, cut into 40 µm sections and treated as described in the Materials and Methods. Note that PKC is, as expected, located in the cytoplasm in unstimulated conditions and selectively translocated to cell-cell contacts upon stimulation. PKC is excluded from cell-matrix contacts as attested by the laminin immunostaining.

View larger version (62K): [in a new window]   Fig. 3. Subcellular localisation of PKC (A) and PKC (B) in unstimulated and TPA or TRH stimulated pituitary glands of transgenic mice exhibiting GFP-GH expressing cells. Pituitary glands were removed from the animals, incubated in HamF10 culture medium containing or not 100 nM TPA or TRH for 1 hour, fixed in paraformaldehyde, cut into 40 µm sections and immunostained with antibodies raised against the and PKC isoforms. Both enzymes are cytoplasmic under unstimulated conditions and selectively translocated to cell-cell contacts of green fluorescent GH cells as well as a subpopulation of GFP-GH-negative cells.

View larger version (92K): [in a new window]   Fig. 4. Subcellular localisation of endogenous PKC and in unstimulated and TPA-stimulated GH3B6 cells. Cells were incubated in the presence of 100 nM TPA for 1 hour and immunostained as described in Materials and methods. PKC and are selectively accumulated at the cell-cell contacts in the presence of TPA.

View larger version (44K): [in a new window]   Fig. 5. The GD(E)E motif located in the V3 region of both PKC and is critical but not sufficient for the targeting to cell-cell contacts. GFP-tagged wild-type and mutated PKC, and were transiently transfected in GH3B6 cells. Cells were stimulated or not with 100 nM TPA. A selective translocation to cell-cell contacts was only observed for the wild-type PKC and upon TPA stimulation. This targeting was abolished when the GD(E)E motif within the V3 region of both isoforms was mutated (mutants PKC-D294G and PKC-E374G). The presence of this motif is however not sufficient to target PKC to cell-cell contacts.

View larger version (37K): [in a new window]   Fig. 6. The V3 variable region does not contain the full information to target PKC to cell-cell contacts. (A) Although C1-GFP is, as expected (Oancea et al., 1998), translocated to the plasma membrane upon TPA stimulation, the (C1+V3)-GFP wild-type and mutated constructs do not translocate in similar conditions. (B) the N terminus of PKC, including the variable V1 and the pseudosubstrate regions, restores targeting to the plasma membrane with no selectivity for cell-cell contacts.

View larger version (52K): [in a new window]   Fig. 7. Cell-cell contact targeting may be controlled through the PKC C2-V3 region. (A) although the deletion of the V1 domain abolishes PKC translocation, the selective targeting to cell-cell contacts is restored in the absence of both V1 and the pseudosubstrate domain. Thus, the PKC N terminus including the V1 and the pseudosubstrate domains is not required for PKC targeting to cell-cell contacts. (B) A further deletion of the catalytic domain does not affect the targeting to cell-cell contacts. A comparison of the (C1-V3)-GFP and the (C1+V3)-GFP (see Fig. 6A) subcellular locations suggests that the C2 region may also be required for the selectivity of targeting to cell-cell contacts.

View larger version (39K): [in a new window]   Fig. 8. Hypothetical model of the subcellular targeting of PKC. PKC translocation to the plasma membrane is regulated through at least two distinct mechanisms: a selective targeting to cell-cell contacts mediated through the C2-V3 region (and at least the V3 domain of ) and a non-selective targeting to the entire plasma membrane involving the V1 and the pseudosubstrate domains. In both cases the translocation to the plasma membrane may require the release of PKC from a cytoplasmic anchoring protein, which should be a RICK. This protein, involved in the cytoplasmic sequestration of PKC, would interact with the C2-V3 region and possibly also with the pseudosubstrate region. Once released from binding to the cytoplasmic anchoring protein, C2-V3 would bind another protein, a RACK, whose expression or ability to bind to PKC is induced by cell-cell contacts. This protein may be either a shuttle or a cargo/anchoring protein that selectively targets PKC to cell-cell contacts upon stimulation. RICK, receptor for inactive c-kinase; RACK, receptor for active c-kinase; Ad J, adherens junctions; ECM, extracellular matrix; DAG, diacylglycerol.