Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.     

Membrane effects of aminoglycoside antibiotics measured in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate

The mechanism of membrane disturbance by aminoglycoside antibiotics was investigated in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS). Liposomes of PC and different anionic phospholipids (1:1 to 15:1 molar ratios) were challenged with aminoglycosides in the presence of low (1 microM) and high (3 mM) concentrations of calcium. Liposomes containing PIP2 showed the greatest drug-induced changes in ANS fluorescence in the presence of high and low concentrations of calcium and at all PC:PIP2 molar ratios tested. Liposomes containing other anionic phospholipids (PS, PI and PIP) were not reactive toward aminoglycosides in the presence of 3 mM calcium or when the ratio of PC to anionic lipid was increased to 10:1. The aminoglycoside-induced changes of ANS fluorescence were not due to any changes in the emission spectrum of ANS, nor to changes in quantum yield, nor to a change in the binding affinity of ANS. It is concluded that a specific aminoglycoside-PIP2 interaction results in phase separation of PC and PIP2 and thus increases the number of available ANS binding sites in PC:PIP2 liposomes.     

Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (adseverin) by some phospholipids

A 74-kDa protein (adseverin) derived from adrenal medulla severs actin filaments and nucleates actin polymerization in a Ca2(+)-dependent manner but does not form an EGTA-resistant complex with actin monomers, which is different from the gelsolin-actin interaction. The dissociation of gelsolin-actin complexes by phosphatidylinositol 4,5-bisphosphate (PIP2) and the inhibitory effect on actin filament severing by gelsolin was recently reported. This study shows that the activity of adseverin is inhibited not only by PIP2 but also by some common phospholipids including phosphatidylinositol (PI) and phosphatidylserine (PS). Other phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no effect. The addition of PC or PE to PI diminished the inhibitory effect of PI. Triton X-100 and neomycin were also found effective in suppressing the effect of PI, suggesting that the arrangement of polar head groups is important in exerting the inhibitory effect. Ca2(+)-dependent binding of adseverin to PS liposomes but not to PC or PE liposomes was observed by a centrifugation assay.     

Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells

Nerve growth factor induces neurite process formation in pheochromacytoma (PC12) cells and causes the parallel increase in levels of the microtubule-associated proteins, tau and MAP1, as well as increases in tubulin levels. Mechanisms to insure balanced accumulation of microtubule proteins and make their levels highly responsive to nerve growth factor were investigated. The effects on tau, MAP1, and tubulin are due to changes in protein synthesis rates, which for tau and tubulin we could show are due in part to changes in the mRNA levels. Whereas tubulin shows feedback regulation to modulate synthesis up or down, tau protein synthesis is not affected in a straightforward way by microtubule polymerization and depolymerization. The degradation of tau, MAP1, and both tubulin polypeptides, however, are stimulated by microtubule depolymerization caused by colchicine, or nerve growth factor removal. Combined feedback on synthesis and stability make tubulin levels highly responsive to assembly states. In addition, the linkage of tau and MAP1 turnover with the state of microtubule polymerization amplifies any change in their rate of synthesis, since tau and MAP1 promote microtubule polymerization. This linkage lends itself to rapid changes in the state of the system in response to nerve growth factor.     

Effect of N-ethylmaleimide on rat liver phosphatidylcholine-specific and non-specific transfer protein activities: its dependence on donor liposome composition

Phosphatidylcholine exchange between liposomes and mitochondria catalyzed by rat liver phosphatidylcholine transfer protein is strongly stimulated by N-ethylmaleimide (NEM) when PC/PI (molar ratio, 4:1) donor liposomes are used. In the presence of PC/PE or PC liposomes the exchange activity by this protein is unaffected. In the same experimental conditions, the activity of rat liver non-specific transfer protein is always stimulated by N-ethylmaleimide with all the types of liposomes tested in the order PC/PI greater than PC/PE greater than PC. Since the effect of NEM depends on the type of liposomes used and appears to be similar for both phospholipid transfer proteins, the possibility that their mode of action implies the formation of a ternary complex should be considered. As far as non-specific transfer protein is concerned, its interaction could vary depending on the nature of the exchanging membranes. Data are also presented indicating that when the two transfer proteins are together their activity is additive, therefore suggesting a specific role in phospholipid biomembrane assembly for each of them.     

Nucleotide torsional flexibility in solution and the use of the lanthanides as nuclear-magnetic-resonance conformation probes. The case of adenosine 5''-monophosphate

Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.     

The periodic association of MAP2 with brain microtubules in vitro

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.     

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.     

Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly.

The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.     

TTLL7 is a mammalian beta-tubulin polyglutamylase required for growth of MAP2-positive neurites

Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for alpha-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.     

Interaction of the water-soluble protein aprotinin with liposomes: gel-filtration, turbidity studies, and 31P NMR studies

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; 31P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV-aprotinin complexes was determined. Lipid-protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. 31P NMR spectroscopy of the MLV-aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Different assembly properties of cod, bovine, and rat brain microtubules.

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.     

Evaluation of novel microtubules interfering agents myoseverin, tubulyzine and E2GG in primary cultures of rat hepatocytes

We investigated the effects of novel microtubules interfering agents (MIAs) in primary cultures of rat hepatocytes. Cells were treated for 24 h with a known compound colchicine and newly synthesized derivatives myoseverin, tubulyzine, and E2GG. We examined the effects of MIAs on microtubules network integrity and on the polymerization capability of isolated tubulin. All tested MIAs inhibited microtubules assembly with the following IC(50) values: tubulyzine (4.4 + or - 0.9 micromol/l), myoseverin (7.0 + or - 0.8 micromol/l), E2GG (16 + or - 2 micromol/l), colchicine (2.0 + or - 0.4 micromol/l). The potency of MIAs to perturb microtubular network integrity (monitored by immune-histochemistry) increased in the order tubulyzine < myoseverin < E2GG < colchicine. We described recently deleterious effects of MIAs on the expression of drug metabolizing enzymes, including CYP1A1. Here we observed inhibitory effects of novel MIAs on dioxin-inducible expression of CYP1A1 mRNA in rat hepatocytes. We conclude that novel MIAs exert analogical biological response as classical MIAs such as colchicine or nocodazole. This further supports the hypothesis that tubulin is the primordial target of MIAs within the cell and that perturbation of microtubules dynamics and/or integrity triggers the biological effects described here.     

Influence of microtubule-associated proteins on the differential effects of paclitaxel and docetaxel

Microtubules are complex structures arising in part from the polymerization of tubulin dimers. Tubulin binds to a wide range of drugs which have been used as probes for tubulin conformation and assembly properties. There is some evidence that taxol and taxotere have differing effects on tubulin conformation. Previous work has shown that MAP2 and Tau, although they both induce microtubule assembly, have qualitatively different effects on tubulin's behavior. Since most microtubulesin vivo are likely to be associated with MAPs, we decided to characterize the differential effects of MAP2, Tau, taxol, and taxotere on tubulin polymerization with the aim of understanding the mechanisms through which these agents stimulate microtubule assembly. Furthermore, the inhibitive effect of calcium has been used to elucidate the ability of the two drugs to force tubulin assembly. These observations suggest that docetaxel, in addition to its greater efficiency in tubulin assembly, may have the capacity to differently alter certain classes of microtubules. Tau and MAP2 accessory proteins may represent important cofactors modulating the effects of taxoids.     

Inhibition of microtubule formation by metabotropic glutamate receptors

Activation of glutamate receptors is known to alter the biophysical state of the cytoskeleton of neurons in the developing brain. In this study, we examined the ability of G protein-coupled metabotropic glutamate receptors (mGluRs) to inhibit the formation of processes induced by the expression of the microtubule-associated protein MAP2c. The infection of insect MG-1 cells with a recombinant baculovirus (BV) encoding MAP2c induced the formation of fine filamentous processes. The binding of MAPs to tubulin promotes tubulin polymerization and the formation of microtubules. Co-infection with BVs for the phosphoinositide (PI)-linked mGluR1a or mGluR1b receptor subtypes inhibited the formation of processes induced by MAP2c, whereas co-infection with BVs encoding the mGluR4a or mGluR4b subtypes that couple to adenylyl cyclase did not inhibit the formation of processes. The biochemical pathways responsible for producing the inhibitory effect of mGluR1 were investigated. Inhibitors of protein kinase C, calcium/calmodulin-dependent kinase, and protein tyrosine kinases did not block the inhibitory effect of mGluR1a. The calcium chelator BAPTA and the calcium depletor thapsigargin also did not affect the ability of mGluR1a to inhibit process formation. In contrast, inhibitors of phospholipase C reversed the effect of mGluR1 on process formation, suggesting that one or more metabolites in the PI pathway were responsible for the inhibitory effect. These findings indicate that PIs generated by activation of mGluRs inhibit the binding of MAPs to tubulin and reduce tubulin polymerization and microtubule stability.     

An antioxidant-like action for non-peroxidisable phospholipids using ferrous iron as a peroxidation initiator

The degradation of phospholipids containing polyunsaturated fatty acids, termed peroxidation, poses a constant challenge to membranes lipid composition and function. Phospholipids with saturated (e.g. PC 16:0/16:0) and monounsaturated fatty acids (e.g. PC 16:0/18:1) are some of the most common phospholipids found in membranes and are generally not peroxidisable. The present experiments show that these non-peroxidisable phospholipids, when present in liposomes with peroxidisable phospholipids (i.e. those containing polyunsaturated fatty acids) such as PC 16:0/18:2 and Soy PC, produce an inhibitory effect on rates of peroxidation induced by ferrous-iron. This inhibitory effect acts to extend the duration of the lag phase by several-fold. If present in natural systems, this action could enhance the capacity of conventional antioxidant mechanisms in membranes. The results of this preliminary work suggest that non-peroxidisable phospholipids may exert an antioxidant-like action in membranes.     

Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.