Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium.
PMA causes rapid down-modulation of CD4 molecules on murine immature thymocytes, human PBL, and CD4-positive human tumor cell lines, but not on murine peripheral lymphocytes. The mechanisms of phorbol ester-induced down modulation of CD4 molecules, however, have not been elucidated. To determine how PMA down-modulates CD4 expression by T lymphocytes, we studied the ability of inhibitors of protein kinase C, calmodulin, actin, and tubulin to block PMA-induced modulation of CD4 in several murine and human cell types. We also tested the ability of intracellular and extracellular calcium chelators to block CD4 internalization. There was marked variability in the degree of PMA-induced down-modulation of CD4 among various cell types. The effects of PMA on CD4 expression were greater for murine thymocytes, for human PBL, and for the human lymphoblastic leukemia cell line, MOLT-3, than for any of the other cell types studied. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked phosphorylation but not internalization of CD4 molecules induced by PMA. Therefore, phosphorylation of CD4 molecules by protein kinase C is not required for the internalization of the molecules. Internalization was blocked by both inhibitors of calmodulin, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and trifluoperazine. PMA-induced internalization of CD4 was blocked by Quin-2 AM, which chelates intracellular calcium. EGTA, which chelates extracellular calcium, did not block internalization. Inhibitors of actin or tubulin did not block internalization. These results suggest that PMA-induced modulation of CD4 can occur in the absence of phosphorylation of the CD4 molecules and is calmodulin and intracellular calcium dependent.
Bigby
M
M
Department of Pathology, Massachusetts General Hospital, Boston.
Wang
P
P
Fierro
J F
JF
Sy
M S
MS
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000199:Actins / Q000502:physiology; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D003571:Cytochalasin B / Q000494:pharmacology; D003593:Cytoplasm / Q000502:physiology; D003703:Demecolcine / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014404:Tubulin / Q000502:physiology
D000199:Actins; D015704:CD4 Antigens; D002147:Calmodulin; D007546:Isoquinolines; D010879:Piperazines; D014404:Tubulin; D003571:Cytochalasin B; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D003703:Demecolcine