Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts

We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.

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