Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer

Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.     

Resolution of cyclic AMP stimulated protein kinase from polymerization-purified brain microtubules.

Polymerization-deploymerization purified microtubules from mouse brain contain, in addition to tubulin, several minor proteins, including protein kinase activity. The protein kinase copurifies with microtubules in constant proportion to tubulin through two, three, or four cycles of polymerization; it can be resolved from tubulin by gel filtration chromatography and has an apparent molecular weight of 280,000. Its activity is stimulated 7-fold by cyclic AMP, and resembles the soluble brain protein kinase described by Miyamoto $\text{et al.}$ (1). The microtubule preparation serves as an endogenous substrate for this protein kinase; both 6S and 30S tubulin are substrates for phosphorylation to the extent of about 0.10 ± 0.05 moles/mole.     

Interaction of maize (Zea mays) protein phosphatase 2A with tubulin

Immunological and biochemical evidence has been obtained for an interaction of maize protein phosphatase 2A (PP2A) holoenzyme with tubulin. Tubulin co-purifies with maize seedling PP2A. Affinity chromatography of the maize PP2A preparation on immobilized tubulin revealed two peaks of phosphorylase alpha phosphatase activity. In one of the peaks, the catalytic (C) and constant regulatory (A) subunits of PP2A were identified by Western blotting. The subunits (C and A) of PP2A were co-immunoprecipitated from maize seedlings homogenate by an anti-alpha-tubulin antibody. The interaction of plant PP2A with tubulin indicates a possible role of reversible protein phosphorylation in the dynamic structure of plant cytoskeleton.     

Calmodulin-dependent protein kinase (kinase II) which is involved in the activation of tryptophan 5-monooxygenase catalyzes phosphorylation of tubulin

Tubulin was shown to be an endogenous substrate of the calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase [T. Yamauchi and H. Fujisawa (1983) Eur. J. Biochem.132, 15–21]. Serine and threonine were identified as the phosphate acceptor amino acids of tubulin. The V_max of the phosphorylation of tubulin and the apparent K_m value for tubulin of calmodulin-dependent protein kinase II were 89 nmol phosphate transferred min^?1 mg kinase II^?1 and 1.7 μm, respectively. The maximum ³²P incorporation into tubulin was 0.18 mol P_i/mol α-tubulin and 0.13 mol P_i/mol β-tubulin. The phosphorylation of tubulin was decreased by the denaturation of tubulin. The phosphorylation of tubulin by kinase II did not affect the assembly of microtubules.     

Purification of bovine adrenocortical and brain tubulin. A comparative study

Microtubules from the cow adrenal cortex and brain were purified by three cycles of the temperature-dependent polymerization-depolymerization procedure. Whereas tubulin comprised approximately 8--10% of soluble brain protein, it comprised only 0.5-1.0% of the soluble adrenocortical protein. The partially purified tubulin from both sources gave similar results in the following studies: (1) [3H]colchicine binding examined by Scatchard analysis revealed an apparent Ka of 1 . 10(6) M-1 and a colchicine/tubulin molar binding ratio of 0.4-0.6; (2) tyrosylation studies using a specific tubulin-tyrosine ligase (which adds a tyrosine residue to the C-terminal glutamate or glutamine of the alpha-chain) in conjunction with carboxypeptidase A (which recovers the tyrosine) and (3) amino acid analysis. Examination of protein bands, in addition to the tubulin doublet of 55 000 molecular weight, on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a difference between the two tubulin preparations. The adrenocortical preparation had protein bands corresponding to apparent molecular weight of 36 000, 60 000, and 68 000. In contrast the brain preparation had only proteins of molecular weights greater than 200 000 (these bands were absent in all adrenal preparations). It would thus appear that if proteins which copurify with tubulin through repeated cycles of polymerization-depolymerization play a role in either microtubule formation or function there is a distinct difference between neural and non-neural tissue.     

Enzymatic acitivity in tubulin preparations: cyclic-AMP dependent protein kinase activity of brain microtubule protein.

Cyclic-AMP stimulates phosphorylation of Hf1 and Hf2b histone fractions and of protamine by tubulin preparations. Lyophilization of the tubulin removes the requirement for cyclic-AMP; the kinase is fully active in the absence of cyclic nucleotide. The casein kinase activity of tubulin is independent of cyclic-AMP. Only cyclic-IMP can replace cyclic-AMP at the low concentrations at which the cyclic-AMP is effective. 5′-AMP inhibits basal levels of Hf 1 histone phosphorylation. Several attempts have been made to separate the kinase activity from tubulin. Both tubulin and kinase activity are precipitated by vinblastine. Mg²⁺ precipitation of tubulin inactivates the kinase; there is no evidence that the precipitation step removes the kinase from tubulin. Tubulin which has been assembled into microtubules, collected by centrifugation and disassembled, retains its kinase activity. Kinase activity and tubulin co-elute from DEAE-cellulose. Taken together, these experiments demonstrate that tubulin, prepared by fractionation with ammonium sulfate followed by elution from DEAE Sephadex with 0-8 M KC1 and concentration by reprecipitation with ammonium sulfate has a closely associated, cyclic-AMP dependent protein kinase activity, which may be intrinsic to the tubulin peptides themselves.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.     

Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin

Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.     

Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP): identification of sites targeted by different kinases

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K(d) = 2.5-2.7 microM) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.     

Interaction of tubulin with octyl glucoside and deoxycholate. 1. Binding and hydrodynamic studies

Tubulin purified from calf brain cytoplasm, normally a compact water-soluble dimer, is able to interact with the mild detergents octyl glucoside (a minimum of 60 detergent molecules) and deoxycholate (95 +/- 8 molecules). Binding is cooperative and approaches saturation below the critical micelle concentration of the amphiphiles. Binding is accompanied by a quenching of the intrinsic protein fluorescence, but no spectral shape changes indicating denaturation such as in the case of sodium dodecyl sulfate are observed. Glycerol, which is known to be preferentially excluded from the tubulin domain and to favor the folded and associated forms of this protein, inhibits the binding of the mild detergents. Octyl glucoside induces a rapidly equilibrating tubulin self-association reaction characterized by a bimodal sedimentation velocity profile with boundaries at approximately 5 and 12 S. Full dissociation of this detergent restores the normal sedimentation behavior to 90% of the protein. Binding of deoxycholate slows the sedimentation velocity of tubulin from s(0)20,w = 5.6 +/- 0.2 S to s(0)20,w = 4.8 +/- 0.3 S. Measurements of the molecular weight of the tubulin-deoxycholate complex indicate an increase from 100,000 to 143,000 +/- 5,000. The diffusion rate consistently decreases from (5.3 +/- 0.5) X 10(-7) to (3.8 +/- 0.2) X 10(-7) cm2 S-1. This is most simply interpreted as an expansion of the undissociated tubulin dimer upon detergent binding (a change in the frictional ratio, f/f min, from 1.35 to 1.86). It is concluded that tubulin shows a reversible transition between the water-soluble state and amphipathic detergent-bound forms which constitute a model system of tubulin-membrane interactions.     

Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in beta-tubulin.

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta III-tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta I-tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.     

Stability of microtubule protein over the pH range: 6.9--9.5.

The pH stability range of a microtubule protein preparation has been investigated between 6.9 and 9.5. Microtubule protein was exposed to various pH values in this range and then returned to pH 6.9. The appearance of microtubules as verified by electron microscopy and sedimentation analysis under polymerizing conditions was taken as an indication of a conformationally stable protein. Between pH 6.9 and pH 8.0 the loss in the ability to form microtubules was found to be reversible, at pH 8.2 it was partially reversible, above pH 8.2 it was irreversible. Tubulin and the microtubule-associated protein fraction were separately exposed to high pH. It was observed that tubulin exposed to high pH can still form microtubules in the presence of untreated microtubule-associated protein. On the other hand, microtubule-associated protein exposed to high pH could not initiate microtubule assembly with untreated tubulin. It was concluded from these observations that the loss in the ability of a microtubule protein preparation to assemble at high pH is due to a change in the microtubule-associated protein fraction and that tubulin is conformationally stable even after exposure to pH 9.5.     

Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.     

Separation of Assembly-Competent Tubulin from Brain Microtubule Protein Preparations Using a Fast-Performance Liquid Chromatography Procedure

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-morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, 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 KCl with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., less than 1 versus 60 mU/mg/min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmol/mg/min 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 nucleotide/tubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ligand- and microtubule assembly-induced GTPase activity of purified calf brain tubulin

Calf brain tubulin purified by means of ammonium sulfate fractionation, ion-exchange chromatography, and MgCl₂ precipitation contains a low level of Mg²⁺ -dependent GTPase activity, the protein preparation being essentially homogeneous according to conventional procedures. Tubulin was freed from this possibly contaminant enzyme activity by Sephacryl S300 gel chromatography. Soluble tubulin itself showed a ligand-induced Mg²⁺-dependent GTPase activity in the presence of colchicine, but not of tropolone methyl ether, podophyllotoxin, or vinblastine. Tubulin also hydrolyzed GTP when assembling into microtubules. This reaction proceeded in a nonlinear fashion and was suppressed together with microtubule assembly by lowering the protein concentration under the critical concentration, adequately modifying assembly buffer conditions, or using Ca²⁺, tropolone methyl ether, podophyllotoxin, or vinblastine. The number of molecules of GTP hydrolyzed per molecule of tubulin polymerized was estimated to vary between 0.9 and 2.1, depending on whether morpholineethanesulfonate or phosphate assembly buffers were employed.     

Tubulin does not bind glycerol irreversibly

Tubulin incorporates radioactivity when incubated with preparations of [¹⁴C]-glycerol, but the material that binds to tubulin is a contaminant rather than glycerol itself. The apparent binding of glycerol by tubulin was not stoichiometric, nor was it significantly affected by dilution of the radioactive glycerol preparation with unlabelled glycerol. Radioactive glycerol purified by prior exposure to protein was an order of magnitude less effective in binding to tubulin than unpurified glycerol. Cytochrome-C and bovine plasma albumin, as well as tubulin, incorporated radioactivity from isotopically labelled glycerol. The amount of radioactivity incorporated into tubulin was unaffected by assembly-disassembly.     

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of ATP in rat cerebral cortex

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4°C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A.70, 765–768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (Pl) fraction and cold-soluble tubulin in the supernatant (Sl) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of ³²P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of ³²P.     

Protein-kinase-C-catalyzed phosphorylation of the microtubule-binding domain of microtubule-associated protein 2 inhibits its ability to induce tubulin polymerization

It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C.     

Legionella micdadei protein kinase catalyzes phosphorylation of tubulin and phosphatidylinositol.

Legionella micdadei, a pathogen which enters into host phagocyte phagolysosomal structures, contains at least two protein kinases. We have purified to homogeneity the predominant, nucleotide-independent protein kinase and examined its ability to catalyze the transfer of phosphate from ATP to acceptors in human neutrophils. The L. micdadei protein kinase catalyzed the phosphorylation of proteins of 11.5, 14, 19, 23, 28, 34, and 38 kilodaltons (kDa) present in a Triton X-100 extract of neutrophil membranes and of 11.5, 13.5, 25, and 38 kDa in the neutrophil cytosol. Tubulin was a good substrate for the L. micdadei protein kinase in vitro. The bacterial kinase also catalyzed the phosphorylation of phosphatidylinositol (PI) at about half the rate at which histones were phosphorylated; phosphatidylinositol-4-phosphate (PIP) was not phosphorylated by the kinase. The PI kinase activity of the L. micdadei enzyme was optimum at pH 7.0, and the divalent cation requirement was satisfied best by Mg2+ and Ca2+. The maximum rate of PI phosphorylation was obtained with 0.6 mM PI; in the presence of MgCl2 (10 mM), the Km for PI was 0.9 mM and the Km for ATP was 1.5 mM. The detergents octyl-beta-D-glucoside (10 to 20 mM) and Triton X-100 (0.5%) stimulated kinase activity twofold when PI was the phosphate acceptor; however, only octyl glucoside stimulated histone kinase activity. Various membrane phospholipids inhibited PI kinase activity. The most potent phospholipid inhibitor was the product of the PI kinase reaction, PIP, which at a 0.6 mM concentration inhibited both PI and tubulin phosphorylation by 80%. The inhibition of kinase activity by PIP when histone served as the acceptor was noncompetitive in character. The L. micdadei kinase also phosphorylated PI in intact. (3H)inositol-labeled neutrophils. The PI kinase and histone kinase activities of teh L. micdadei kinase copurified and cofucused (pI, 5.8) when subjected to isoelectric focusing, suggesting that the two enzymatic activities reside in a single protein.     

The role of tubulin in the mitochondrial metabolism and arrangement in muscle cells

Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.