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Increased protein tyrosine phosphorylation in apoptotic neural cell death due to microtubule perturbations

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Abstract

The microtubule-perturbing drugs colchicine and taxol have been found to induce apoptosis in a CNS neuronal cell line. Apoptosis in drug-treated rat B103 neuroblastoma cells was evident in characteristic morphological changes, internucleosomal DNA fragmentation, and loss of nuclear content. Since colchicine and taxol have opposite actions on microtubule integrity, disruption of the active turnover of the microtubule network appears to be a crucial step for apoptosis to occur. It has been suggested that the basis for apoptosis by these drugs derives from their known block of the cell cycle at G2/M, but this does not appear the sole reason as both colchicine and taxol were able to evoke high levels of apoptosis in cells differentiated by Bt2cAMP or serum withdrawal. Further tests of cellular consequences of microtubule perturbation revealed a specific impact on signal transduction involving protein tyrosine phosphorylation. Immunoprecipitation with antibodies against tyrosine phosphorylated proteins showed a striking increase in the phosphorylation of a Triton-insoluble ~90 kDa protein, roughly concurrent with the onset of internucleosomal DNA fragmentation. Cycloheximide and genistein significantly reduced cell death and blocked appearance of the ~90 kDa tyrosine phosphorylated protein. Data suggest the hypothesis that signal transduction leading to apoptosis can be triggered by anomalous microtubule turnover and that the mechanism involves tyrosine phosphorylation of a ~90 kDa Triton-resistant protein.

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Abbreviations

MTs:

microtubules

Bt2cAMP:

dibutyrl cyclic AMP

VEC-DIC:

Video-enhanced-contrast-differential-interference-contrast

DMEM:

Dulbecco’s modified Eagle media

DMSO:

dimethylsulfoxide

D-PBS:

Dulbecco’s Phosphate buffered saline

HRP:

horse radish peroxidase

PMSF:

phenylmethylsulfonyl fluoride

PI:

propidium iodide

NFT:

neurofibrillary tangles

ADDLs:

Amyloid-peta derived diffusible ligands

AD:

Alzheimer’s disease

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Authors and Affiliations

  1. Dept. of Neurobiology and Physiology, Northwestern University, 2153 N. Campus Dr., 60208, Evanston, IL

    Brett A. Chromy, Mary P. Lambert & William L. Klein

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  1. Brett A. Chromy
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  2. Mary P. Lambert
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  3. William L. Klein
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Correspondence to William L. Klein.

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Chromy, B.A., Lambert, M.P. & Klein, W.L. Increased protein tyrosine phosphorylation in apoptotic neural cell death due to microtubule perturbations. neurotox res 2, 357–372 (2000). https://doi.org/10.1007/BF03033343

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