A synthetic peptide homologous to retroviral transmembrane envelope proteins depresses protein kinase C mediated lymphocyte proliferation and directly inactivated protein kinase C: a potential mechanism for immunosuppression.
CKS-17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS-17-BSA). To define its site of action, we determined if CKS-17 conjugated to human serum albumin (CKS-17-HSA) could block the direct activation of lymphocytes by phorbol-12-myristate-13-acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiC8). CKS-17-HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose-dependent manner with up to 88% inhibition occurring with 15 microM CKS-17-HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 microM CKS-17-HSA. Based on these findings we investigated the effect of CKS-17-HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS-17-HSA caused a dose-dependent enzyme inhibition with a concentration giving half-maximal inhibition (IC50) of ca.3 microM and greater than 95% inhibition at 15 microM CKS-17-HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS-17-HSA did not inhibit the activity of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PK-A) nor the calcium and phospholipid-independent form of PKC (PK-M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA-stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS-17-HSA may be due to the direct inactivation of PKC.
Kadota
J
J
Second Department of Internal Medicine, Nagasaki University School of Medicine.
Cianciolo
G J
GJ
Snyderman
R
R
D000595:Amino Acid Sequence; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D006801:Humans; D007165:Immunosuppression Therapy; D036341:Intercellular Signaling Peptides and Proteins; D008213:Lymphocyte Activation / Q000187:drug effects*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D015682:Retroviridae Proteins, Oncogenic / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000494:pharmacology*
D004075:Diglycerides; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D015682:Retroviridae Proteins, Oncogenic; D014759:Viral Envelope Proteins; C044295:1,2-dioctanoylglycerol; C053692:CKS 17; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate