Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.
Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor alpha, platelet-activating factor, phorbol ester (phorbol 12-myristate 13-acetate), and calcium ionophore A23187 are able to increase the level of tyrosine phosphorylation of different protein substrates, as demonstrated by Western blotting with anti-phosphotyrosine antibody (anti-PY). A protein of 41 kDa (p41) consistently showed more intense reactivity to anti-PY than controls. Blots treated with anti-PY, stripped of the antibody, and reblotted with microtubule-associated protein kinase (MAPK, p42MAPK) antibody show only one band. The molecular mass of that band exactly matches that of p41. MAPK-reactive protein is present in control and stimulated cells, although the intensity of the band is greater in the latter. GM-CSF-stimulated phosphorylation of p41 is time- and dose-dependent. Anti-MAPK antibody detects a single band of 41 kDa, whose intensity increases with time of incubation and concentration of the agonist. Thus, the anti-MAPK antibody appears to react better to the phosphorylated form of p41 from GM-CSF-stimulated cells than to the dephosphorylated form. The p41 and MAPK proteins are localized in the cytosol. Finally, MAPK immunoprecipitates were probed with anti-PY in Western blots and a band of 41 kDa was found. In summary, these results suggest that this 41-kDa protein in neutrophils that is tyrosine phosphorylated in response to GM-CSF and other stimuli is MAPK. Its phosphorylation may represent an early and crucial signal associated with the GM-CSF neutrophil stimulation cascade.
Gomez-Cambronero
J
J
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
Huang
C K
CK
Gomez-Cambronero
T M
TM
Waterman
W H
WH
Becker
E L
EL
Sha'afi
R I
RI
D000595:Amino Acid Sequence; D000906:Antibodies; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; 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 / Q000276:immunology; D010750:Phosphoproteins / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood*; D011994:Recombinant Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine
D000906:Antibodies; D001798:Blood Proteins; D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate