Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide, fMet-Leu-Phe.
Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-Phe (fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin alpha-subunits, as measured by 14C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact microtubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of alpha-tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells.
Rothwell
S W
SW
Department of Hematology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
Nath
J
J
Wright
D G
DG
D000595:Amino Acid Sequence; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D015151:Immunoblotting; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D014404:Tubulin / Q000097:blood / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D014404:Tubulin; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine