Chemotactic peptides induce phosphorylation and activation of MEK-1 in human neutrophils.
Extracellular signal-regulated kinase (Erk) (mitogen-activated protein (MAP) kinase) is rapidly activated when neutrophils are stimulated. Several isoforms of MAP/Erk kinase (MEK), a kinase capable of phosphorylating and activating Erk, have been identified, but their distribution and differential roles in leukocytes are unknown. We studied the effect of chemotactic stimulation on MEK-1, using isoform-specific antibodies. MEK-1 was found to be phosphorylated on serine and threonine residues in unstimulated human neutrophils. Stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced serine/threonine phosphorylation of MEK-1, while reducing its electrophoretic mobility. MEK-1 activity, measured as autophosphorylation or as phosphorylation of a glutathione S-transferase-Erk fusion protein, was undetectable in unstimulated cells but became evident after treatment with chemoattractant. Phosphorylation and activation of MEK-1 were rapid and transient, peaking after 1-2 min and returning to base line by 10 min. Experiments using electropermeabilized cells indicated that elevation of cytosolic Ca2+ is not required for activation of MEK-1 by fMLP. Moreover, MEK-1 was not stimulated by either platelet-activating factor or thapsigargin, which increase Ca2+ to levels comparable with those attained in chemoattractant-activated cells. In contrast, activation of MEK-1 was induced by phorbol esters, and the stimulatory effect of fMLP was blocked by an antagonist of protein kinase C. Stimulation of MEK-1 was also blocked by concentrations of erbstatin that prevent the fMLP-induced accumulation of tyrosine-phosphorylated proteins. The data suggest that MEK-1 is largely responsible for the activation of Erk in chemoattractant-stimulated neutrophils and that protein kinase C and/or tyrosine kinases mediate this effect, whereas elevated cytosolic Ca2+ is not essential.
Grinstein
S
S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
Butler
J R
JR
Furuya
W
W
L'Allemain
G
G
Downey
G P
GP
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D007527:Isoenzymes; D011994:Recombinant Proteins; D013729:Terpenes; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium