Phosphorylation of components of the ER translocation site

In many eukaryotic cells, protein secretion is regulated by extracellular s ignalling molecules giving rise to increased intracellular Ca2+ and activat ion of kinases and phosphatases. To test whether components involved in the first step of secretion, the translocation of proteins across the endoplas mic reticulum (ER) membrane, are regulated by Ca2+-dependent phosphorylatio n and dephosphorylation, we have investigated the effect of Ca2+ on kinases associated with the rough ER. Using purified rough microsomes from dog pan creas we found that Ca2+-dependent isoforms of protein kinase C (PKC) are a ssociated with the rough ER and phosphorylate essential components of the p rotein translocation machinery. Phosphorylation of microsomal proteins by P KCs increased protein translocation efficiency in vitro. We also found that proteins of the translocation machinery became phosphorylated in intact ce lls. This suggests a further level of regulation of protein translocation a cross the ER membrane.