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

The alpha isoform of protein kinase C (PKC alpha) is a ubiquitous protein k inase, which, upon activation, translocates rapidly from the cytoplasm to t he plasma membrane. To follow this translocation, PKC alpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably ex pressed in baby hamster kidney cells overexpressing the muscarinic type 1 r eceptor. Addition of the agonist carbamylcholine triggered the onset of tra nslocation within 1 s. Half-maximal and maximal translocation occurred afte r about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within abou t 45 s. A high-resolution study showed an almost homogeneous cytoplasmic di stribution of tagged PKC alpha. in unstimulated cells and virtually complet e translocation to the plasma membrane in response to the phorbol ester, PM A. 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 agonis t. At low agonist concentration, a small increase in [Ca2+](i) was observed , without detectable translocation of PkC alpha. In contrast, PMA induced t ranslocation of PKC alpha without any increase in [Ca2+](i). Neither cytoch alasin D nor colcemid influenced the distribution or calcium-dependent tran slocation 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 i ts cytoplasmic localization to the plasma membrane.