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

Abstract

The α isoform of protein kinase C (PKCα) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCα 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 PKCα 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 PKCα 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 PKCα. In contrast, PMA induced translocation of PKCα without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCα, indicating that PKCα translocation may be independent of both actin filaments and microtubules. The time course of PKCα translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.