Irreversible incorporation of L-dopa into the C-terminus of α-tubulin inhibits binding of molecular motor KIF5B to microtubules and alters mitochondrial traffic along the axon.

L-dopa is the most effective drug used to date for management of Parkinson's disease symptoms. Unfortunately, long-term administration of L-dopa often results in development of motor disorders, including dyskinesias. Despite extensive research on L-dopa-induced dyskinesia, its pathogenesis remains poorly understood. We demonstrated previously that L-dopa can be post-translationally incorporated into the C-terminus of α-tubulin in living cells. In the present study, we investigated the effect of the presence of L-dopa-tubulin-enriched microtubules on mitochondrial traffic mediated by molecular motor KIF5B. Using biochemical approaches in combination with experiments on neuronal cell lines and mouse hippocampal primary cultures, we demonstrated that L-dopa incorporation into tubulin is irreversible. Transport of mitochondria along the axon was altered after L-dopa treatment of cells. In L-dopa-treated cells, mitochondria had reduced ability to reach the distal segment of the axon, spent more time in pause, and showed reduced velocity of anterograde movement. KIF5B motor, a member of the kinesin family involved in mitochondrial transport in neurons, showed reduced affinity for Dopa-tubulin-containing microtubules. Our findings, taken together, suggest that tyrosination state of tubulin (and microtubules) is altered by L-dopa incorporation into tubulin; the gradual increase in amount of altered microtubules affects microtubule functioning, impairs mitochondrial traffic and distribution, and this could be relevant in Parkinson's disease patients chronically treated with L-dopa.

Tubulin-Na+, K + -ATPase interaction: Involvement in enzymatic regulation and cellular function.

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na+ ,K + -ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin-NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na + . Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na + and K + , and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na + /K + homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.

Post-translational incorporation of 3,4-dihydroxyphenylalanine into the C terminus of α-tubulin in living cells.

The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal and readdition in a process termed the "detyrosination/tyrosination cycle". We showed in previous studies using soluble rat brain extracts that l-3,4-dihydroxyphenylalanine (l-Dopa) is incorporated into the same site as Tyr. We now demonstrate that l-Dopa incorporation into tubulin also occurs in living cells. We detected such incorporation by determining the "tyrosination state" of tubulin before and after incubation of cells in the presence of l-Dopa. The presence of a tubulin isospecies following l-Dopa incubation that was not recognized by antibodies specific to Tyr- and deTyr-tubulin was presumed to reflect formation of Dopa-tubulin. l-Dopa was identified by HPLC as the C-terminal compound bound to α-tubulin. l-Dopa incorporation into tubulin was observed in Neuro 2A cells and several other cell lines, and was not due to de novo protein biosynthesis. Dopa-tubulin had microtubule-forming capability similar to that of Tyr- and deTyr-tubulin. l-Dopa incorporation into tubulin did not notably alter cell viability, morphology, or proliferation rate. CAD cells (a neuron-like cell line derived from mouse brain) are easily cultured under differentiating and nondifferentiating conditions, and can be treated with l-Dopa. Treatment of CAD cells with l-Dopa and consequent increase in l-Dopa-tubulin resulted in reduction of microtubule dynamics in neurite-like processes.

Post-Translational Incorporation of L-Phenylalanine into the C-Terminus of α-Tubulin as a Possible Cause of Neuronal Dysfunction.

α-Tubulin C-terminus undergoes post-translational, cyclic tyrosination/detyrosination, and L-Phenylalanine (Phe) can be incorporated in place of tyrosine. Using cultured mouse brain-derived cells and an antibody specific to Phe-tubulin, we showed that: (i) Phe incorporation into tubulin is reversible; (ii) such incorporation is not due to de novo synthesis; (iii) the proportion of modified tubulin is significant; (iv) Phe incorporation reduces cell proliferation without affecting cell viability; (v) the rate of neurite retraction declines as level of C-terminal Phe incorporation increases; (vi) this inhibitory effect of Phe on neurite retraction is blocked by the co-presence of tyrosine; (vii) microtubule dynamics is reduced when Phe-tubulin level in cells is high as a result of exogenous Phe addition and returns to normal values when Phe is removed; moreover, microtubule dynamics is also reduced when Phe-tubulin is expressed (plasmid transfection). It is known that Phe levels are greatly elevated in blood of phenylketonuria (PKU) patients. The molecular mechanism underlying the brain dysfunction characteristic of PKU is unknown. Beyond the differences between human and mouse cells, it is conceivable the possibility that Phe incorporation into tubulin is the first event (or among the initial events) in the molecular pathways leading to brain dysfunctions that characterize PKU.

PMCA activity and membrane tubulin affect deformability of erythrocytes from normal and hypertensive human subjects.

Our previous studies demonstrated formation of a complex between acetylated tubulin and brain plasma membrane Ca(2+)-ATPase (PMCA), and the effect of the lipid environment on structure of this complex and on PMCA activity. Deformability of erythrocytes from hypertensive human subjects was reduced by an increase in membrane tubulin content. In the present study, we examined the regulation of PMCA activity by tubulin in normotensive and hypertensive erythrocytes, and the effect of exogenously added diacylglycerol (DAG) and phosphatidic acid (PA) on erythrocyte deformability. Some of the key findings were that: (i) PMCA was associated with tubulin in normotensive and hypertensive erythrocytes, (ii) PMCA enzyme activity was directly correlated with erythrocyte deformability, and (iii) when tubulin was present in the erythrocyte membrane, treatment with DAG or PA led to increased deformability and associated PMCA activity. Taken together, our findings indicate that PMCA activity is involved in deformability of both normotensive and hypertensive erythrocytes. This rheological property of erythrocytes is affected by acetylated tubulin and its lipid environment because both regulate PMCA activity.

Serum-induced neurite retraction in CAD cells--involvement of an ATP-actin retractile system and the lack of microtubule-associated proteins.

Cultured catecholamine-differentiated cells [which lack the microtubule-associated proteins (MAPs): MAP1B, MAP2, Tau, STOP, and Doublecortin] proliferate in the presence of fetal bovine serum, and, in its absence, cease dividing and generate processes similar to the neurites of normal neurons. The reintroduction of serum induces neurite retraction, and proliferation resumes. The neurite retraction process in catecholamine-differentiated cells was partially characterized in this study. Microtubules in the cells were found to be in a highly dynamic state, and tubulin in the microtubules consisted primarily of the tyrosinated and deacetylated isotypes. Increased levels of acetylated or Δ2-tubulin (which are normally absent) did not prevent serum-induced neurite retraction. Treatment of differentiated cells with lysophosphatidic acid or adenosine deaminase induced neurite retraction. Inhibition of Rho-associated protein kinase, ATP depletion and microfilament disruption each (individually) blocked serum-induced neurite retraction, suggesting that an ATP-dependent actomyosin system underlies the mechanism of neurite retraction. Nocodazole treatment induced neurite retraction, but this effect was blocked by pretreatment with the microtubule-stabilizing drug paclitaxel (Taxol). Paclitaxel did not prevent serum-induced or lysophosphatidic acid-induced retraction, suggesting that integrity of microtubules (despite their dynamic state) is necessary to maintain neurite elongation, and that paclitaxel-induced stabilization alone is not sufficient to resist the retraction force induced by serum. Transfection with green fluorescent protein-Tau conferred resistance to retraction caused by serum. We hypothesize that, in normal neurons (cultured or in vivo), MAPs are necessary not only to stabilize microtubules, but also to establish interactions with other cytoskeletal or membrane components to form a stable structure capable of resisting the retraction force.

Quantification of acetylated tubulin.

The acetylation/deacetylation of Lys40 of the α-subunit is an important posttranslational modification undergone by tubulin during the life of a cell. Many previous studies have addressed the physiological role of this acetylation process using various approaches based on changes of acetylated tubulin (AcTubulin) content. In most of these studies, however, the actual amounts of AcTubulin were not known and it was difficult to draw conclusions. We present here a simple method to estimate the percentage of AcTubulin relative to total tubulin. The method is based on acetylation of the tubulin sample with acetic anhydride, Western blotting stained by antiAcTubulin antibody, and comparison of the optical density of the AcTubulin band with that of a corresponding sample that was not chemically acetylated.

A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype.

Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly-disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly-disassembly procedure. The effective inhibitory concentration of TSA was 3 µM in the homogenate and 1 µM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ~64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly-disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly-disassembly kinetics were the same regardless of the degree of acetylation.

High glucose levels induce inhibition of Na,K-ATPase via stimulation of aldose reductase, formation of microtubules and formation of an acetylated tubulin/Na,K-ATPase complex.

Our previous studies demonstrated that acetylated tubulin forms a complex with Na(+),K(+)-ATPase and thereby inhibits its enzyme activity in cultured COS and CAD cells. The enzyme activity was restored by treatment of cells with l-glutamate, which caused dissociation of the acetylated tubulin/Na(+),K(+)-ATPase complex. Addition of glucose, but not elimination of glutamate, led to re-formation of the complex and inhibition of the Na(+),K(+)-ATPase activity. The purpose of the present study was to elucidate the mechanism underlying this effect of glucose. We found that exposure of cells to high glucose concentrations induced: (a) microtubule formation; (b) activation of aldose reductase by the microtubules; (c) association of tubulin with membrane; (d) formation of the acetylated tubulin/Na(+),K(+)-ATPase complex and consequent inhibition of enzyme activity. Exposure of cells to sorbitol caused similar effects. Studies on erythrocytes from diabetic patients and on tissues containing insulin-insensitive glucose transporters gave similar results. Na(+),K(+)-ATPase activity was >50% lower and membrane-associated tubulin content was >200% higher in erythrocyte membranes from diabetic patients as compared with normal subjects. Immunoprecipitation analysis showed that acetylated tubulin was a constituent of a complex with Na(+),K(+)-ATPase in erythrocyte membranes from diabetic patients. Based on these findings, we propose a mechanism whereby glucose triggers a synergistic effect of tubulin and sorbitol, leading to activation of aldose reductase, microtubule formation, and consequent Na(+),K(+)-ATPase inhibition.

Involvement of membrane tubulin in erythrocyte deformability and blood pressure.

OBJECTIVE: To test the hypothesis that erythrocyte deformability is influenced by changes in the content of membrane tubulin (Mem-tub). METHODS AND RESULTS: Human erythrocytes contain tubulin distributed in three pools (membrane, sedimentable, soluble). Erythrocytes from hypertensive humans have a higher proportion of Mem-tub. Increased Mem-tub content in hypertensive patients was correlated with decreased erythrocyte deformability. Treatment of erythrocytes from normotensive individuals with taxol increased Mem-tub content and reduced deformability, whereas treatment of hypertensive patients erythrocytes with nocodazole had the opposite effect. In-vivo experiments with rats were performed to examine the possible relationship between Mem-tub content, erythrocyte deformability, and blood pressure. Spontaneously hypertensive rats (SHRs) showed lower erythrocyte deformability than normotensive Wistar rats. During the development of hypertension in SHR, tubulin in erythrocytes is translocated to the membrane, and this process is correlated with decreased deformability. In-vivo treatment (intraperitoneal injection) of SHR with nocodazole decreased Mem-tub content, increased erythrocyte deformability, and decreased blood pressure, whereas treatment of Wistar rats with taxol had the opposite effects. CONCLUSION: These findings indicate that increased Mem-tub content contributes to reduced erythrocyte deformability in hypertensive animals.

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na+, K+-ATPase activity.

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na(+), K(+)-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.

Lack of stabilized microtubules as a result of the absence of major maps in CAD cells does not preclude neurite formation.

In many laboratories, the requirement of microtubule-associated proteins (MAPs) and the stabilization of microtubules for the elongation of neurites has been intensively investigated, with controversial results being obtained. We have observed that the neurite microtubules of Cath.a-differentiated (CAD) cells, a mouse brain derived cell, are highly dynamic structures, and so we analyzed several aspects of the cytoskeleton to investigate the molecular causes of this phenomenon. Microtubules and microfilaments were present in proportions similar to those found in brain tissue and were distributed similarly to those in normal neurons in culture. Neurofilaments were also present. Analysis of tubulin isospecies originating from post-translational modifications revealed an increased amount of tyrosinated tubulin, a diminished amount of the detyrosinated form and a lack of the Delta2 form. This tyrosination pattern is in agreement with highly dynamic microtubules. Using western blot analyses with specific antibodies, we found that CAD cells do not express several MAPs such as MAP1b, MAP2, Tau, doublecortin, and stable-tubule-only-peptide. The presence of the genes corresponding to these MAPs was verified. The absence of the corresponding mRNAs confirmed the lack of expression of these proteins. The exception was Tau, whose mRNA was present. Among the several MAPs investigated, LIS1 was the only one to be expressed in CAD cells. In addition, we determined that neurites of CAD cells form and elongate at the same rate as processes in a primary culture of hippocampal neurons. Treatment with nocodazol precluded the formation of neurites, and induced the retraction of previously formed neurites. We conclude that the formation and elongation of neurites, at least in CAD cells, are dependent on microtubule integrity but not on their stabilization or the presence of MAPs.

Acetylated tubulin associates with the fifth cytoplasmic domain of Na(+)/K(+)-ATPase: possible anchorage site of microtubules to the plasma membrane.

We showed previously that NKA (Na(+)/K(+)-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubule-plasma membrane anchorage site through an interaction between acetylated tubulin and CD5.

Submembraneous microtubule cytoskeleton: regulation of ATPases by interaction with acetylated tubulin.

The ATP-hydrolysing enzymes (Na(+),K(+))-, H(+)- and Ca(2+)-ATPase are integral membrane proteins that play important roles in the exchange of ions and nutrients between the exterior and interior of cells, and are involved in signal transduction pathways. Activity of these ATPases is regulated by several specific effectors. Here, we review the regulation of these P-type ATPases by a common effector, acetylated tubulin, which interacts with them and inhibits their enzyme activity. The presence of an acetyl group on Lys40 of alpha-tubulin is a requirement for the interaction. Stimulation of enzyme activity by different effectors involves the dissociation of tubulin/ATPase complexes. In cultured cells, acetylated tubulin associated with ATPase appears to be a constituent of microtubules. Stabilization of microtubules by taxol blocks association/dissociation of the complex. Membrane ATPases may function as anchorage sites for microtubules.

Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex.

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex. Here, we report that in plasma membrane vesicles isolated from brain synaptosomes, another P-type ATPase, plasma membrane Ca(2+)-ATPase (PMCA), undergoes enzyme activity regulation by its association/dissociation with acetylated tubulin. The presence of acetylated tubulin/PMCA complex in membrane vesicles was demonstrated by analyzing the behavior of acetylated tubulin in a detergent partition, and by immunoprecipitation experiments. PMCA is known to be stimulated by ethanol and calmodulin at physiological concentrations. We found that treatment of plasma membrane vesicles with these reagents induced dissociation of the complex, with a concomitant restoration of enzyme activity. Conversely, incubation of vesicles with exogenous tubulin induced the association of acetylated tubulin with PMCA, and the inhibition of enzyme activity. These findings indicate that activation of synaptosomal PMCA by ethanol and calmodulin involves dissociation of the acetylated tubulin/PMCA complex. This regulatory mechanism was shown to also operate in living cells.

The effect of early surgical treatment of traumatic spine injuries on patient mortality.

INTRODUCTION: The ideal timing of spinal fixation is controversial. A recent study showed that early spine fixation reduced morbidity and resource utilization. We previously noted a trend toward higher mortality in patients undergoing early spinal fixation. This study was done to analyze whether the timing of spinal fixation had a significant effect on mortality. METHODS: The registry of our Level I trauma program was queried for all patients with at least one spinal vertebral injury. Anatomic and physiologic variables included age, initial Glasgow Coma Scale score, systolic blood pressure, heart rate, and Injury Severity Score. Outcome was evaluated in terms of ventilator days, intensive care unit length of stay, hospital length of stay (HLOS), and mortality. Patients were stratified by day of spinal operative fixation as early when done within 48 hours and late when done after 48 hours. Data were analyzed using chi and an unpaired t test, accepting p < 0.05 as significant. RESULTS: Three hundred sixty-one patients between January 1988 and February 2003 required operative spinal fixation (158 early, within 48 hours vs. 203 late, beyond 48 hours). There was no significant difference between the two groups except mortality, which was significantly higher in the early group (7.6 vs. 2.5%; p = 0.0257), and HLOS, which was significantly shorter in the early group (14.42 vs. 17.64 days; p = 0.025). CONCLUSION: Spinal fixation within 48 hours after vertebral fractures and dislocations appears to increase mortality despite similar anatomic and physiologic parameters in the later operative group. Incomplete resuscitation of patients before surgery may have contributed to this result. The shorter HLOS may have been because of the higher number of early deaths. Prospective studies to identify the optimal timing of spinal fixation and the reason for these outcome differences are warranted.

Tubulin must be acetylated in order to form a complex with membrane Na(+),K (+)-ATPase and to inhibit its enzyme activity.

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.

Activation of the plasma membrane H-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H(+)-ATPase-acetylated tubulin complex.

In the yeast Saccharomyces cerevisiae, plasma membrane H(+)-ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H(+)-ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H(+)-ATPase-tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H(+)-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H(+)-pumping activity. The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H(+)-ATPase) immunoglobulins indicated a physical interaction between H(+)-ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H(+)-ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H(+)-ATPase-tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H(+)-ATPase activity by acetylated tubulin and concomitant increase of the H(+)-ATP ase-tubulin complex.

Regulation of acetylated tubulin/Na+,K+-ATPase interaction by L-glutamate in non-neural cells: involvement of microtubules.

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na+,K+-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes with L-glutamate induces dissociation of the acetylated tubulin/Na+,K+-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells. As in the case of astrocytes, the effect of L-glutamate is mediated by its transporters and not by specific receptors. In COS cells, the effect of L-glutamate was reversed by its elimination from culture medium, provided that d-glucose was present. The effect of L-glutamate was not observed when Na+ was replaced by K+ in the incubation medium. The ionophore monensin, in the presence of Na+, had the same effect as L-glutamate. Treatment of cells with taxol prevented the dissociating effect of L-glutamate or monensin. Nocodazole treatment of intact cells or isolated membranes dissociated the acetylated tubulin/Na+,K+-ATPase complex. The dissociating effect of nocodazol does not require Na+. These results indicate a close functional relationship among Na+,K+-ATPase, microtubules, and L-glutamate transporters, and a possible role in cell signaling pathways.

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.