Epidermoid carcinoma-derived antimicrobial peptide (ECAP) inhibits phosphorylation by protein kinases in vitro

Animal peptide antibiotics are thought to mediate their cytotoxic and growt h inhibitory action on bacteria, fungi, and cancer cells through a membrane -targeted mechanism. Although the membrane interactions of the peptide anti biotics and their penetration through the membranes have been studied in se veral models, the precise chain of events leading to cell death or growth a rrest is not established yet. In this study we used in vitro kinase assays followed by imaging analyses to examine the effect of human cationic antimi crobial peptide ECAP on the activity of the protein kinases. We report that HPLC-grade ECAP is responsible for inhibition of EGFR autophosphorylation in plasma membrane fractions obtained from A-431 cells. The activity of ECA P is concentration dependent with a half-inhibitory concentration in the ra nge of 0.1-0.2 mum. Marked decrease in autophosphorylation of immunoprecipi tated non-receptor protein kinases belonging to different families, namely PKC mu, Lyn and Syk, is observed in the presence of as little as 0.2 mum of the peptide. Among the examined nonreceptor protein kinases PKC mu was the most sensitive to the inhibitory action of ECAP, whereas Syk was inhibited least of all. ECAP exerted no detectable cytotoxicity on non-nucleate anim al cells at concentrations up to 3 pm. The capability of ECAP to inhibit pr otein kinases at concentrations, that are at least 10 fold lower than antib acterial and cytotoxic ones, suggests that the protein kinases are possible intracellular targets for antimicrobial peptides. We suppose that inhibiti on of the protein kinases may provide a mechanism for the action of cationi c antimicrobial peptides on host cells including tumour cells. Copyright (C ) 2001 John Wiley & Sons, Ltd.