Receptor-mediated activation of multiple serine/threonine kinases in human leukocytes.
Activation of the microbicidal response of phagocytes requires cytosolic ATP and is associated with extensive protein phosphorylation, suggesting the involvement of protein kinases in the signal transduction cascade. An in vitro renaturation assay was used to identify the protein kinase(s) activated by chemoattractants in human blood neutrophils. Four distinct kinases were activated by the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine with molecular masses of 72, 65, 49, and 41 kDa (designated PK72, PK65, PK49, and PK41, respectively). PK72 and PK65 were activated very rapidly (5-15 s), yet transiently. By comparison, PK49 and PK41 responded in a slower, more sustained manner. Treatment of extracts of activated cells with alkaline phosphatase reverted the stimulation of the kinases, suggesting that phosphorylation is the post-translational modification that underlies activation of the kinases. Stimulation of PK72 and PK65 by chemoattractant was independent of calcium and protein kinase C. In contrast, elevation of cytosolic free calcium levels was sufficient and appeared to be necessary for full activation of PK49 and PK41. While phorbol esters can mimic the effects of formyl-methionyl-leucyl-phenylalanine on PK49 and PK41, inhibition of protein kinase C by staurosporine did not prevent the receptor-mediated activation of these kinases. PK41 most likely corresponds to the Erk-1 isoform of mitogen-activated protein (MAP) kinase. Accordingly, PK41 effectively phosphorylated myelin basic protein, known to be a good substrate for Erk-1. The electrophoretic mobility of PK49 is similar to that of MAP kinase-kinase (MAP/Erk kinase). However, immunoprecipitation experiments indicated that PK49 is not MAP/Erk kinase. The identity of this and other kinases remains to be defined, but possible candidates are discussed. In addition to autophosphorylating, PK72, PK65, and PK41 were shown to effectively phosphorylate exogenous substrates. These kinases may therefore play a role in signal transduction during stimulation by chemoattractants.
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
S
S
Grinstein
W
W
Furuya
J R
JR
Butler
J
J
Tseng
D001483:Base Sequence; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000097:blood; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D007527:Isoenzymes; D010455:Peptides; D010748:Phosphopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013729:Terpenes; D004533:Egtazic Acid; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium