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Ionic requirements and subcellular localization of tubulin tyrosinolation in human polymorphonuclear leukocytes
Abstract:
We previously reported a specific stimulation of polymorphonuclear leukocyte (PMN) tubulin tyrosinolation as induced by the peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-phe) and the Ca2+ ionophore A23187 that is coupled to the NADPH oxidase-mediated stimulation of the PMN respiratory burst. The present study demonstrates that the presence of extracellular Ca2+ is necessary for fmet-leu-phe- and A23187-induced stimulation of PMN tubulin tyrosinolation, as indicated by the complete inhibition of the response by the addition of 1 mM EGTA to the extracellular medium. Methoxyverapamil (10(-5) M), a putative calcium channel blocker, completely inhibited the fmet-leu-phe-induced stimulation of tubulin tyrosinolation in PMN, but did not inhibit the A23187-induced response. Moreover, the calmodulin-binding drugs, trifluoperazine, fluphenazine, or chlorpromazine, at concentrations of 1 to 10 microM, caused significant inhibition of fmet-leu-phe- or A23187-induced stimulation of tubulin tyrosinolation. In related studies, enzymatic [14C]-tyrosinolation in isolated subcellular fractions of PMN revealed the presence of native tubulin in PMN fractions that were enriched in plasma membranes, the specific granules, or the azurophil granules. Most interestingly, tubulin tyrosine ligase (ligase), primarily a cytoplasmic enzyme, was detected in association with the PMN azurophil granule-rich fraction. Immunoautoradiography with the alpha-tubulin antibody YL 1/2 of isolated PMN subcellular fractions demonstrated a preferential stimulation of tyrosinolation of tubulin associated with the plasma membrane-rich fraction of fmet-leu-phe-stimulated cells. A significant stimulation was also observed in the cytoplasmic tubulin fraction. Consistent with the findings of in vitro tyrosinolation studies with PMN subcellular fractions, tyrosinolated tubulin was detected in the azurophil granule-enriched fractions isolated from both resting and fmet-leu-phe-stimulated cells. The antibody YL 1/2, which reacts with tyrosinolated alpha-tubulin and not with the detyrosinolated form, showed significant cross-reaction with several nontubulin PMN proteins.
Insights
Extracellular calcium is essential for N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-phe) and A23187-induced polymorphonuclear leukocyte (PMN) tubulin tyrosinolation. Calmodulin inhibitors and calcium channel blockers differentially affect these responses, suggesting distinct signaling pathways.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) play a crucial role in immune responses.
- Tubulin tyrosinolation is a post-translational modification of alpha-tubulin, influencing microtubule dynamics.
- N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-phe) and Ca2+ ionophore A23187 are known stimuli for PMN activation and respiratory burst.
Purpose of the Study:
- To investigate the role of extracellular calcium in fmet-leu-phe- and A23187-induced PMN tubulin tyrosinolation.
- To explore the involvement of calcium channels and calmodulin in these signaling pathways.
- To determine the subcellular localization of tubulin and tubulin tyrosine ligase in PMNs.
Main Methods:
- PMN isolation and stimulation with fmet-leu-phe or A23187 in the presence or absence of extracellular Ca2+ (EGTA).
- Inhibition studies using methoxyverapamil (calcium channel blocker) and calmodulin-binding drugs (trifluoperazine, fluphenazine, chlorpromazine).
- Enzymatic [14C]-tyrosinolation assays and immunoautoradiography with anti-alpha-tubulin antibody (YL 1/2) on isolated PMN subcellular fractions.
Main Results:
- Extracellular Ca2+ is indispensable for both fmet-leu-phe- and A23187-induced PMN tubulin tyrosinolation.
- Methoxyverapamil inhibited fmet-leu-phe-induced tyrosinolation but not A23187-induced tyrosinolation.
- Calmodulin inhibitors significantly suppressed both fmet-leu-phe- and A23187-induced tubulin tyrosinolation.
- Native tubulin and tubulin tyrosine ligase were found in various PMN subcellular fractions, including azurophil granules.
- Tyrosinolation of tubulin associated with the plasma membrane-rich fraction was preferentially stimulated by fmet-leu-phe.
Conclusions:
- Extracellular calcium influx is a critical requirement for PMN tubulin tyrosinolation stimulated by both chemoattractants and ionophores.
- Distinct signaling mechanisms involving calcium channels and calmodulin mediate the tubulin tyrosinolation response to different stimuli.
- Tyrosinolated tubulin is present in PMN subcellular compartments, including azurophil granules and plasma membrane fractions, suggesting roles in cellular functions.