Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.
A cycle of posttranslational modification of alpha-tubulin has previously been described in higher eukaryotes, in which a C-terminal tyrosine residue is removed and replaced by two complementary cytoplasmic enzymes. The activity of the detyrosinating enzyme, tubulin carboxypeptidase (TCP), and its potential for regulating the level of detyrosinated (Glu) subunits in microtubules (MTs) is of great interest, since TCP catalyzes the primary modification of tubulin and since the level of Glu alpha-tubulin in MTs increases during a variety of differentiative and morphogenetic events. As a first step in examining the role of TCP in cellular morphogenesis, it was necessary to develop an assay for TCP with sufficient sensitivity and specificity to detect TCP activity during these events. Unlike previously described assays for TCP, ours makes use of the affinity TCP exhibits for MTs. NGF-induced neurite outgrowth in PC-12 cells was accompanied by a moderate (approximately 2-fold) increase in TCP activity, while myogenesis of L6 cells resulted in an almost insignificant decrease in activity. Measurements of TCP activity during differentiation were correlated with the level of extract Tyr tubulin, which increased (approximately 37%) during neurite outgrowth and was unchanged during myogenic differentiation. Our results suggest that TCP activity is regulated relative to its substrate, Tyr tubulin, and that changes in MT dynamics, rather than enzymatic activities, are the primary determinants of MT posttranslational modification state during differentiation. In addition, the assay we have devised for TCP and the characterization of TCP during differentiation may allow the future delineation of the mechanism(s) of regulation of TCP and the role this enzyme plays in modulating MT function during differentiation.
Webster
D R
DR
Department of Cell Biology & Anatomy, College of Physicians & Surgeons, Columbia University, New York, New York 10032.
Modesti
N M
NM
Bulinski
J C
JC
D000310:Adrenal Gland Neoplasms; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D002417:Cattle; D002454:Cell Differentiation; D003593:Cytoplasm / Q000201:enzymology*; D051379:Mice; D008870:Microtubules / Q000502:physiology*; D009024:Morphogenesis; D009132:Muscles / Q000648:ultrastructure*; D009414:Nerve Growth Factors; D016501:Neurites / Q000502:physiology* / Q000648:ultrastructure; D010673:Pheochromocytoma; D051381:Rats; D013552:Swine; D014404:Tubulin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D009414:Nerve Growth Factors; D014404:Tubulin; D014443:Tyrosine; D002268:Carboxypeptidases; C023212:tyrosyltubulin carboxypeptidase