Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol.
Receptor-linked activation of phospholipase D has been demonstrated recently in a variety of intact cell types including granulocytes, but little is known about the enzyme, its cofactor requirements, and regulation. Using [3H]alkyllysophosphatidylcholine to prelable an endogenous phosphatidylcholine substrate pool in conjunction with transphosphatidylation using ethanol to generate labeled phosphatidylethanol, we demonstrated a novel phospholipase D activity in neutrophil subcellular fractions. Guanosine 5'-O-3-(thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) activated both phosphatidic acid generation and transphosphatidylation. Activity using both activators required the presence of not only plasma membrane but also cytosol, and proteolytic and thermal inactivation demonstrated the requirement for protein factors in both fractions. Using both stimuli, activity increased with increasing cytosol concentration. Product formation was approximately linear for about 10 min with PMA and 30 min with GTP gamma S, and both activators resulted in the total hydrolysis of up to 10% of the labeled phosphatidylcholine. The activity using both activators showed similar broad neutral pH optima, and both required the presence of micromolar levels of calcium, which by itself failed to activate at concentrations up to 1 mM. At low micromolar concentrations of nucleotides, activation was specific for guanine nucleotides and showed a specificity of GTP gamma S greater than guanyl-5'-yl imidodiphosphate greater than GTP, with no effect of GDP and GMP or adenine nucleotides, consistent with the participation of a guanine nucleotide regulatory protein. PMA activation was dependent on the presence of ATP, in particular when dialyzed cytosol was used, and was inhibited by about 50% by staurosporine, supporting a role for protein kinase C. However, purified protein kinase C failed to substitute for cytosol, implicating an additional cytosolic factor(s) in this response. These results indicate that the granulocytic phospholipase D pathway is a complex system that is regulated by at least two activation pathways, each comprised of components in two subcellular compartments.
Olson
S C
SC
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
Bowman
E P
EP
Lambeth
J D
JD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology; D020404:Glycerophospholipids; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D019311:Staurosporine; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000470:Alkaloids; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D011506:Proteins; C051521:phosphatidylethanol; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000431:Ethanol; D000255:Adenosine Triphosphate; D010739:Phospholipase D; D019311:Staurosporine; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium