Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis.
Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.
Ball
R L
RL
Department of Microbiology, University of Texas Medical Branch, Galveston.
Albrecht
T
T
Thompson
W C
WC
James
O
O
Carney
D H
DH
D000818:Animals; D001666:Binding Sites, Antibody / Q000187:drug effects; D002478:Cells, Cultured / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D004815:Epidermal Growth Factor; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate