Simultaneous visualization of the translocation of protein kinase Cα-green fluorescent protein hybrids and intracellular calcium concentrations

The alpha isoform of protein kinase C (PKC alpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKC alpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKC alpha. in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+](i)) and PKC alpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+](i) was observed, without detectable translocation of PkC alpha. In contrast, PMA induced translocation of PKC alpha without any increase in [Ca2+](i). Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKC alpha, indicating that PKC alpha translocation may be independent of both actin filaments and microtubules. The time course of PKC alpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.