The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes.
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.
Sironi
J J
JJ
Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Argentina.
Barra
H S
HS
Arce
C A
CA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002268:Carboxypeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008870:Microtubules / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D002268:Carboxypeptidases; C023212:tyrosyltubulin carboxypeptidase