Cellular uptake and metabolism of myristoylated N-terminal peptides of PKA C-subunit.

Abstract

Peptides are, in general, unable passively to traverse the plasma membrane. However, the attachment of a fatty acyl group to a previously non-permeant peptide is usually sufficient to overcome the cellular permeability barrier and to allow such a peptide access to the cell interior [ 11 .This principle has been exploited in the development of N-myristoylated inhibitors for protein kinases, based on the sequences of inhibitory (pseudosubstrate) peptides, which are effective in intact cells [ 1,2]. Numerous instances exist where a protein-protein interaction is specifically antagonized by a peptide having sequence identity to that of a contact domain on one of the interacting partners. Studies of such antagonism in intact cells have usually been precluded by the inability of the peptide in question to cross the cell membrane. The catalytic (C-) subunit of PKA is N-myristoylated [3] and it is now recognized that this covalent modification is shared in common among a large number of otherwise diverse proteins involved in intracellular signalling [4]. In well-analysed examples of myristoylated proteins, the function of the N-myristoyl modification is to predispose them to interact with apolar domains either on cellular membranes or on other proteins 151. Although no such targeting role has been definitively ascribed to the myristoylated N-terminus of PKA C-subunit, the possibility has not been rigorously excluded. A powerful demonstration of such a role would emerge if myristoylated peptides related to the N-terminal sequence of C-subunit could be shown to disrupt PKA-mediated functions in intrt cells. As a prelude to such studies, we have examined the ability of the myristoylated Cal-12 peptide (myrCal-12) to enter fibroblasts, compared with that of the non-myristoylated analogue and we have investigated whether de-myristoylation occurs at a rate sufficient to confound the results of prospective functional disruption experiments. Peptides Cal-12 and myrCal-12 were synthesised, with an additional tyr at the C-terminus, purified, analysed and finally radioiodinated using conventional methods of peptide chemistry. Sheep 1' skin fibroblasts were grown to 80% confluence in replicate 6-well plates. Normal growth medium was then supplemented with one or other of the peptides at a concentration of 50pg/ml (approx. 5OpCi/pg). Samples of growth medium were removed at l O m i intervals. Cell monolayers were rapidly washed in several changes of ice-cold PBS then lysed. Medium and lysate samples were analysed for the presence of labelled peptide by reversed phase hplc. Blank experiments, using empty wells, were necessary to control for the timedependent sequestration of the peptides (particularly myrCal-12) onto the tissueculture plastic-ware. Lysates were similarly prepared from fibroblasts that had not been exposed to labelled peptide. These were used to test whether demyristoylase activity was associated with these cells, using [3H]myrCal-12 as a substrate peptide and monitoring release of ['Hlmyristic acid by reversed phase hplc. Cal-12 peptide was not taken-up from the medium by fibroblasts in the course: of a l h incubation and was unaffected by the presence of the cell monolayer. Contrastingly, myr 50% of this labelled peptide remaining in the growth medium had undergone some modification resulting in a change to its peptide peptide c1-12 myrcl-12 125-1