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Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed., (Copyright 2000 Academic Press.)

Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from alpha-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction. We ruled out, by using several control experiments, the possibility that this result was due to contamination of the isolated cytoskeletons by non-associated proteins contained in the detergent fraction or to an artifact in vitro during the extraction procedure. The associated carboxypeptidase activity showed characteristics similar to those of brain tubulin carboxypeptidase and different from those of pancreatic carboxypeptidase A. In comparison with cultures at confluence, those at low cell density contained small (if any) amounts of carboxypeptidase activity associated with microtubules. In addition, the enzyme was shown to be associated only with cold-labile microtubules. The tubulin carboxypeptidase/microtubule association was also demonstrated in Chinese hamster ovary, NIH 3T3 and PC12 cells. Interestingly, this association was not observed in cultured embryonic brain cells. Our results demonstrate that tubulin carboxypeptidase is indeed associated with microtubules in living cells. Furthermore, the findings that this association occurs with a subset of microtubules and that its magnitude depends on the degree of confluence of the cell culture indicate that it could be part of the mechanism that regulates the tyrosination state of microtubules.

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the alpha-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75-78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.