Background: B inhibits P of several proteins by different kinases, and thus may interfere with cell regulatory mechanisms. B inhibition of substrate P is prevented by poly-Lys. Subdomain II on protein kinases contains an invariant Lys. Hypothesis: a synthetic peptide consisting of the Lys-containing region of subdomain II will prevent B inhibition of substrate P by APK, while peptides in which Lys is substituted with arginine or alanine will not have this effect. Methods: The Lys-containing peptide had this sequence: Gly-Asp-His-Tyr-Ala-Met-Lys-Ile-Leu-Glu. In two other peptides alanine or arginine were substituted for Lys. Phospholemman [10, 30, or 50μM] was incubated at 30°C, pH 7.4 for 3 min in microtiter plates containing (final conc.): 0.1 μM BSA, 0.2 mM ATP (with 1-4 μCi[γ-32P]ATP/assay), and 2.5 nM PKA. Reactions were performed± B [50 μM] and one of the peptides [300 μM] and were stopped by adding 0.2 M EDTA. The solution was transferred to SpinZyme separation units and washed ×3 with 75 mM phosphoric acid. Radioactivity was measured as Cerenkov radiation. Results: B inhibition of substrate P was partially prevented by the Lys-containing peptide (seefigure). In the absence of B the Lys-peptide by itself did not have any effect on substrate P. The non-Lys peptides did not have this effect (F=2.31, p= 0.097, data not shown). Discussion: We have previously suggested that binding of B to Lys may play a role in B inhibition of peptide P, and thus in the mediation of B toxicity. The present results support this hypothesis, and suggest that binding of B to Lys on subdomain II of protein kinases may be involved. The Lys-containing peptide copies part of the primary structure of subdomain II, and the secondary and tertiary structures may well be different from the original. We speculate that this may, at least in part, explain why the B effect is not completely prevented by the Lys-containing peptide.

figure 1

Figure 1