Isolation of rat liver microtubule-associated proteins. Evidence for a family of microtubule-associated proteins with molecular mass of around 200,000 which distribute widely among mammalian cells

Heat-stable microtubule-associated proteins (MAPs) were isolated from rat liver crude extract. The most prominent species showed a molecular mass close to that of bovine adrenal 190-kDa MAP (Kotani, S., Murofushi, H., Maekawa, S., Sato, C., and Sakai, H. (1986) Eur. J. Biochem. 156, 23-29), termed rat 190-kDa MAP. Immunological studies with antiserum against the rat 190-kDa MAP showed that this MAP exists in a variety of rat cells and tissues. The characteristics of the rat 190-kDa MAP, including molecular mass, heat stability, and distribution pattern, were very similar to those of bovine adrenal 190-kDa MAP. However, one-dimensional peptide mapping revealed considerable difference, and there is little mutual immunological cross-reactivity. We also identified in mouse neuroblastoma Neuro 2a cells a MAP of around 200 kDa which is considered to be MAP4 (Parysek, L. M., Asnes, C.F., and Olmsted, J.B. (1984) J. Cell Biol. 99, 1309-1315). MAP4 was slightly immunoreactive to both anti-(rat 190-kDa MAP) antiserum and anti-(bovine 190-kDa MAP) antiserum. Taking these results together, we conclude that mammalian tissues ubiquitously contain heat-stable MAPs of 200 kDa and that these 200-kDa MAPs should be considered as species-specific homologues.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.     

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Identification of a 100 kD protein associated with microtubules, intermediate filaments and coated vesicles in cultured cells

We have obtained several hybridoma clones producing antibodies to microtubule-associated proteins (MAPs) from bovine brain. Interaction of one of these antibodies, named RN 17, with cultured cells was studied by indirect immunofluorescence and immunoelectron microscopy. RN 17 antibody recognized both high molecular weight (HMW) MAPs, MAP 1 and MAP 2, in immunoblotting reaction with brain microtubules. In lysates of cultured cells, it bound to a protein doublet with a molecular weight of 100 kD. By immunofluorescence microscopy we showed that RN 17 antibody stained cytoplasmic fibrils, mitotic spindles and small particles in the cytoplasm of various cultured cells. The cytoplasmic fibrils were identified as both microtubules and intermediate filaments by double fluorescence microscopy and by their response to colcemid and 0.6 M KCl. This identification was confirmed by immunoelectron microscopy which also showed that the particles stained by RN 17 antibody are coated vesicles. Thus, cultured non-neural cells may contain a novel protein that binds to microtubules, intermediate filaments, and coated vesicles.     

PC12 cells express juvenile microtubule-associated proteins during nerve growth factor-induced neurite outgrowth

Microtubule-associated proteins (MAPs) are believed to play an important role in regulating the growth of neuronal processes. The nerve growth factor-induced differentiation of PC12 pheochromocytoma cells is a widely used tissue culture model for studying this mechanism. We have found that contrary to previous suggestions, the major MAPs of adult brain, MAP1 and MAP2, are minor components of PC12 cells. Instead two novel MAPs characteristic of developing brain, MAP3 and MAP5, are present and increase more than 10-fold after nerve growth factor treatment; the timing of these increases coinciding with the bundling of microtubules and neurite outgrowth. Immunocytochemical staining showed that MAP3 and MAP5 are initially distributed throughout the cytoplasm. Subsequently MAP5 becomes associated with microtubules in both neurites and growth cones but MAP3 distribution remained diffuse. Thus MAP3 and MAP5, which are characteristic of developing neurons in the juvenile brain, are also induced in PC12 cells during neurite outgrowth in culture. In contrast MAP1, which is characteristic of mature neurons, does not increase during PC12 cell differentiation. These results provide evidence that one set of MAPs is expressed during neurite outgrowth and a different set during the maintenance of neuronal form. It also appears that the PC12 system is an appropriate model for studying the active neurite growth phase of neuronal differentiation but not for neuronal maturation.     

Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross- reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross- reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.     

Generation of microtubule stability subclasses by microtubule- associated proteins: implications for the microtubule "dynamic instability" model

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.     

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.     

Rapid phosphorylation of microtubule-associated proteins through distinct mitogenic pathways

Mitogenic stimulation of sparse quiescent Swiss 3T3 cells with serum induces a transient reorganization of microtubules which may be necessary for generation or transduction of the mitogenic signal(s). Recently, several studies have shown that microtubule-associated proteins (MAPs) modulate microtubule-mediated functions in vitro and in vivo. We have analyzed, by two-dimensional electrophoresis, the molecular changes in MAPs associated with microtubules in situ following cell activation. By as early as 15 min after addition of serum, several of the MAPs present in quiescent cells are lost from the assembled microtubule fraction while one additional MAP becomes evident. This new MAP is a phosphoprotein whose appearance is independent of protein synthesis. Four additional MAPs also become phosphorylated, and this phosphorylation is accompanied by a partial redistribution of MAPs into the unassembled soluble fraction. Stimulation of cells with purified platelet-derived growth factor or phorbol tumor promoter, a direct activator of protein kinase C, also induces phosphorylation of the same MAPs and DNA synthesis. These results demonstrate that activation of the protein kinase C pathway is sufficient to promote the phosphorylation of MAPs and mitogenesis. However, epidermal growth factor, which does not activate protein kinase C, also stimulates phosphorylation of MAPs and DNA replication. Furthermore, down-regulation of the protein kinase C pathway does not prevent these responses. We conclude that phosphorylation of MAPs and mitogenesis can proceed through protein kinase C-dependent and -independent pathways in 3T3 cells.     

270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon

MAPs (microtubule-associated proteins) were isolated from crayfish walking leg nerves. A major MAP was identified as a high molecular weight protein (270K). This protein co-migrated with mammalian MAP2, stimulated the polymerization of rat brain tubulin into microtubules, and was heat resistant. Rotary shadowing revealed that the 270K MAP is a long thin flexible structure. It formed cross-bridges of fine strands, linking microtubules with each other in vitro. These strands resemble the cross-bridges between microtubules observed in the crayfish axon permeabilized with saponin and quick-frozen, deep-etched. Antibodies against mammalian MAP2 cross-reacted with this crayfish MAP and stained the axoplasm of the walking leg nerves. Thus MAPs, especially the 270K MAP, appear to be a major component of the cross-linking strands between microtubules observed in the crayfish axon.     

Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly-disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (greater than 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.     

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)     

A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs)

The effect of the antimitotic drug taxol on the association of MAPs (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of Taxol at 37 degrees C. at 0 degrees C, and at 37 degrees C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter two conditions. At 37 degrees C and at 0 degrees C, complete assembly of both tubulin and the MAPs was observed in the presence of Taxol. However, at elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAPs. The MAPs could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAPs of high purity from small amounts of biological material. The MAPs could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAPs were prepared from bovine cerebral cortex (gray matter) and from HeLa cells. MAP 1, MAP2, and the tau MAPs, as well as species of Mr = 28,000 and 30,000 (LMW, or low molecular weight, MAPs) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 mol wt HeLa MAPs were isolated from HeLa cells. Microtubules were also prepared for the first time from white matter. All of the MAPs identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the two preparations was a fivefold lower level of MAP 2 relative to tubulin in white matter than in gray. The high molecular weigh MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. These results indicate that MAP 1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.     

Selective stabilization of tau in axons and microtubule-associated protein 2C in cell bodies and dendrites contributes to polarized localization of cytoskeletal proteins in mature neurons

In mature neurons, tau is abundant in axons, whereas microtubule- associated protein 2 (MAP2) and MAP2C are specifically localized in dendrites. Known mechanisms involved in the compartmentalization of these cytoskeletal proteins include the differential localization of mRNA (MAP2 mRNA in dendrites, MAP2C mRNA in cell body, and Tau mRNA in proximal axon revealed by in situ hybridization) (Garner, C.C., R.P. Tucker, and A. Matus. 1988. Nature (Lond.). 336:674-677; Litman, P., J. Barg, L. Rindzooski, and I. Ginzburg. 1993. Neuron. 10:627-638), suppressed transit of MAP2 into axons (revealed by cDNA transfection into neurons) (Kanai, Y., and N. Hirokawa. 1995. Neuron. 14:421-432), and differential turnover of MAP2 in axons vs dendrites (Okabe, S., and N. Hirokawa. 1989. Proc. Natl. Acad. Sci. USA. 86:4127-4131). To investigate whether differential turnover of MAPs contributes to localization of other major MAPs in general, we microinjected biotinylated tau, MAP2C, or MAP2 into mature spinal cord neurons in culture (approximately 3 wk) and then analyzed their fates by antibiotin immunocytochemistry. Initially, each was detected in axons and dendrites, although tau persisted only in axons, whereas MAP2C and MAP2 were restricted to cell bodies and dendrites. Injected MAP2C and MAP2 bound to dendritic microtubules more firmly than to microtubules in axons, while injected tau bound to axonal microtubules more firmly than to microtubules in dendrites. Thus, beyond contributions from mRNA localization and selective axonal transport, compartmentalization of each of the three major MAPs occurs through local differential turnover.     

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.     

A common amino acid sequence in 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly

Previously we reported that chymotryptic fragments of bovine adrenal 190-kDa microtubule-associated proteins (27-kDa fragment) and bovine brain tau (14-kDa fragment) contained microtubule-binding domain (Aizawa, H., Murofushi, H., Kotani, Hisanaga, S., Hirokawa, N., and Sakai, H. (1987) J. Biol. Chem. 262, 3782-3787; Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1988) J. Biol. Chem. 263, 7703-7707). In order to study the structure of microtubule-binding domain of the two microtubule-associated proteins, we analyzed the amino acid sequence of the 27-kDa fragment and compared the sequence with that of the 14-kDa fragment. This revealed that 190-kDa microtubule-associated protein and tau contained at least one common sequence of 20 amino acid residues in their microtubule-binding domains. A synthetic polypeptide corresponding to the common sequence (Lys-Asn-Val-Arg-Ser-Lys-Val-Gly-Ser-Thr-Glu-Asn-Ile-Lys- His-Gln-Pro-Gly-Gly-Gly-Arg-Ala-Lys) was bound to microtubules competitively with the 190-kDa MAP. The apparent dissociation constant (KD) for the binding of the polypeptide to microtubules was estimated to be 1.8 x 10(-4) M, and the maximum binding reached 1.2 mol of the synthetic polypeptide/mol of tubulin dimer. This synthetic polypeptide increased the rate and extent of tubulin polymerization and decreased the critical concentration of tubulin for polymerization. The polypeptide-induced tubulin polymers were morphologically normal microtubules and were disassembled by cold treatment. The common sequence (termed assembly-promoting sequence) was thus identified as the active site of 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly. The reconstitution system of microtubules with this synthetic polypeptide with assembly-promoting sequence may be useful to elucidate detailed molecular mechanism of the promotion of microtubule assembly by microtubule-associated proteins.     

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.     

The novel microtubule-associated protein MAP3 contributes to the in vitro assembly of brain microtubules

MAP3 is a novel microtubule-associated protein found in brain and a variety of other tissues (Huber, G., Alaimo-Beuret, D., and Matus, A. (1985) J. Cell Biol. 100, 496-507). In this study, monoclonal antibodies were used to assess its influence on the polymerization of brain tubulin. When added to unpolymerized brain microtubules, anti-MAP3 IgG produced a dose-related inhibition of subsequent assembly. Under the same circumstances, nonimmune mouse IgG did not influence either the rate or the extent of tubulin polymerization. We also used immobilized antibodies to deplete brain MAPs selectively in either MAP3 or MAP1. MAP3-depleted MAPs showed a reproducible decrease in activity compared to control preparations that had been exposed to immobilized nonimmune IgG. MAP1-depleted MAPs did not differ significantly in performance from the nonimmune treated controls. We conclude that MAP3 contributes to the net assembly of brain microtubules observed in vitro. This may be particularly relevant in neonatal animals where brain MAP3 is more abundant than in the adult.     

Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules

Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.