Molecular strategies for the dominant inhibition of protein kevase C

Abstract

The regulatory (R) domain of PKC alpha fused to glutathione-S-transferase (GST-R alpha) competitively inhibited PKC activity associated with extracts of Y1 mouse adrenocortical tumor cells and the activities of several specific PKC isozymes. GST-R alpha did not inhibit the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase or calmodulin-dependent myosin light chain kinase. GST-R alpha inhibited PKC activities 20 times more potently than did a synthetic peptide corresponding to the pseudosubstrate sequence of PKC alpha. In intact yeast cells, the R domain prevented PKC beta-1-induced inhibition of growth and cytokinesis. These results indicate that the R domain of PKC alpha acts as a specific, dominant inhibitor of PKC activity, and suggest that the PKC alpha R domain may provide a useful genetic tool to assess the roles of PKC in various signal transduction processes.

Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by bovine protein kinase C α expression in Saccharomyces cerevisiae

“…This prevents the hydrolysis of the inducing agent (galactose) and enables the galactose-inducible promoter to function in the presence of the preferred carbon source (glucose). Mammalian PKCs expressed in yeast have functional characteristics similar to those found in mammalian cells (Riedel et al, 1993a,b,c;Goode et al, 1994;Shieh et al, 1996;Parissenti et al, 1996b;Keenan et al, 1997;Parissenti et al, 1999). For example, classical PKC isoenzymes expressed in yeast can be activated by phorbol esters (Riedel et al, 1993a,b).…”

Global Profiles of Gene Expression Induced by Adrenocorticotropin in Y1 Mouse Adrenal Cells

“…Forskolin, a well-characterized activator of most isoforms of adenylyl cyclase including those expressed in the Y1 cell line (43), and the active cAMP analog 8BrcAMP were used to probe the cAMP/protein kinase A pathway. The protein kinase C-dependent signaling cascade was probed using the phorbol ester PMA, a well-characterized activator of most protein kinase C isoforms, including the isoforms (␣ and ⑀) that are most abundant in Y1 adrenal cells (44). Although these reagents are commonly used activators of their respective pathways, each has other activities (45)(46)(47) that prompted the inclusion of several additional criteria to validate their modes of action.…”