Phosphorylation and aggregation of neurotubulin and ‘associated’ protein-kinase (original) (raw)
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Molecular and cellular biochemistry, 1997
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 modification...
THE PHOSPHORYLATION OF BRAIN MICROTUBULAR PROTEINS IN SITU AND IN VITRO
Journal of Neurochemistry, 1976
The phosphorylation of microtubular proteins isolated by reassembly in vitro from slices of guinea-pig cerebral cortex labelled with [32P]orthophosphate was investigated. Under the conditions tested, both and the α and β forms of tubulin contained metabolically-active P which accounted for about one third of the total 32P incorporated into protein; the remaining protein-bound 32P was associated with 3–4 minor high MW components co-purifying with tubulin during two cycles of assembly4sassembly. Microtubular protein prepared in this way contained approx. 0.8 mol of alkali-labile P/mol of tubulin dimer (M.W. 110,000).In vitro studies showed that reassembled microtubular protein preparations catalysed the incorporation of up to 0.55 mol of P/mol of tubulin dimer during incubation with Mg2+ and [γ32P]ATP. The reaction was linear during the first 30 min of incubation at 37°C. Cyclic AMP (10 μm, final concentration) caused a transient increase in the initial rates of tubulin phosphorylation. Little label was incorporated into the minor high M.W. components under these conditions. The in vitro phosphorylation of microtubular protein increased in a non-linear manner with respect to protein concentration: this was in contrast to earlier experiments showing linear kinetics when chromatographically isolated tubulin was tested for intrinsic kinase activity. Isolated microtubular protein preparations bound [3H]GTP, [3H]ATP and to a lesser extent, [3H]cyclic AMP, and exhibited Ca2+-ATPase activity (up to 60pmol Pi released min/mg protein at 37°C).
Journal of Neurochemistry, 1986
Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. Microtubule protein (1 -100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-mor-pho1ino)ethanesulfonic acid, 0.5 mM MgCI2, 1 mM EGTA, 0.3 M KCI, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KC1 with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., < I versus 60 mU/mgl min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmolimglmin of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotideitubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mgiml in the presence of 0.03 mM taxol and 1 mM GTP at 37°C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein. However, it is possible to separate active tubulin-nucleotide rapidly from the major microtubule-associated proteins, enzyme contaminants, and unbound nucleotide with just Mono Q column chromatography. Key Words: Brain tubulin-Microtubule-Neurotubule-Microtubule-associated proteins-HPLC -Fast-performance liquid chromatography. Roychowdhury S. and Gaskin F. Separation of assembly-competent tubulin from brain microtubule protein preparations using a fast-performance liquid chromatography procedure.
Phosphorylation of tubulin by a calmodulin-dependent protein kinase.J. Biol. Chem. 261:10332-10339
Journal of Biological Chemistry
Calmodulin-dependent protein kinase was purified from porcine brain cytosol through sequential steps involving acid precipitation, DEAE-chromatography, and calmodulin-Sepharose chromatography. The purified enzyme contained a major M, 50,000 and a minor M, 60,000 peptide. Porcine brain tubulin was a major substrate for this kinase. Under optimal conditions 2.6 mol of phosphate were incorporated per mol of tubulin. The kinase phosphorylated both tubulin subunits at their carboxyl-terminal region. Limited proteolysis, using trypsin and chymotrypsin, of phosphorylated and unphosphorylated tubulins resulted in different cleavage patterns as determined by peptide mapping. Phosphorylated tubulin was unable to bind to microtubule-associated protein or to polymerize, but regained its assembly capacity after phosphatase treatment.
Journal of Biological Chemistry
Insulin receptor kinase phosphorylated tubulin in an insulin-dependent fashion. Two different populations of phosphotubulin were found. In tubulin dimers containing tyrosine at the carboxyl-terminal of their a subunit, phosphate was incorporated in that residue, and the phosphorylated protein did not assembIe into polymers. In tubulin dimers lacking this tyrosine residue, phosphate was incorporated into different tyrosine residues located in other parts of the molecule, and the phosphoprotein retained its capacity to polymerize.
Association of brain g-tubulins with ab-tubulin dimers
Biochemical …, 2002
g-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by g-tubulin and the regulation of this process are not fully understood. Here we show that there are two g-tubulin forms in brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of high-salt concentration. Both g-tubulins copolymerized with tubulin dimers and multiple g-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against g-tubulin and a-tubulin revealed interactions of both g-tubulin forms with tubulin dimers irrespective of the size of complexes. We suggest that besides small and large g-tubulin complexes, other molecular g-tubulin form(s) exist in brain extracts. Twodimensional electrophoresis revealed multiple charge variants of g-tubulin both in brain extracts and in microtubule protein preparations. Post-translational modification(s) of gtubulins might therefore play an important role in the regulation of microtubule nucleation in neuronal cells.
Molecular and Cellular Biochemistry, 2006
In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na + ,K +-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na + ,K +-ATPase and consequent inhibition of enzyme activity.
Enzymatic Detyrosination of Tubulin Tyrosinated in Rat Brain Slices and Extracts
Journal of Neurochemistry, 1982
Tubulin was tyrosinated in slices and in extracts of brain of rats of 3, 25, and 120 days of age by successive incorporation of [14C]tyrosine and ?HItyrosine, respectively. The release of the incorporated amino acid was measured by using tubulinyl-tyrosine carboxypeptidase, carboxypeptidase A, and tubulin-tyrosine ligase. With the carboxypeptidases no differences in either the rates or the extents of the release of tyrosine between these two differently labeled tubulins were found. Differences were found when the detyrosination was catalyzed by the ligase and these were attributed to a higher inactivation of tubulin labeled in slices than of that labeled in extracts.