Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms.
[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.
O'Flaherty
J T
JT
Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27013.
Jacobson
D P
DP
Redman
J F
JF
Rossi
A G
AG
D001667:Binding, Competitive; D029742:Caenorhabditis elegans Proteins; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D004075:Diglycerides / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D007975:Leukotriene B4 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D011493:Protein Kinase C / Q000097:blood*; D011955:Receptors, Drug / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D010743:Phospholipids; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; C044295:1,2-dioctanoylglycerol; D007975:Leukotriene B4; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D013110:Sphingosine; C005682:safingol; D002118:Calcium