The multiple phosphorylation of the microtubule-associated protein MAP2 controls the MAP2:tubulin interaction

Pre-phosphorylation of the microtubule-associated protein MAP2 with the co-purifying cAMP-independent protein kinase (a) decrease the affinity of MAP2 for taxol-stabilised microtubules, (b) increases the dissociation rate constant for microtubule polymerisation, each of which is dependent upon the level of phosphorylation, but (c) has no effect on the association rate constant. Microtubule assembly has no effect on the kinetics of phosphorylation, whereas phosphorylation of pre-assembled microtubules causes their immediate depolymerisation at a rate which is proportional to the initial rate of phosphorylation. The results suggest that the modulated phosphorylation of MAP2 may regulate microtubule length in vivo.     

GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling

A major determinant of neuronal morphology is the cytoskeleton. And one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of protein kinases and phosphatases present in neurons, including glycogen-synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3beta to analyze possible effects on microtubule stability and on the association of MAP2 with microtubules. We have found that GSK3beta phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Additionally, the formation of microtubule bundles, which is observed after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3beta wild type. Microtubule bundles were not observed in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3beta wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3beta wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein preparations. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.     

The phosphorylation of a putative sperm microtubule-associated protein 2 (MAP2) is uniquely sensitive to regulation.

We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule-associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes.     

A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2)

In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consists of two structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures. MAP 2 is known to be specifically phosphorylated as the result of a protein kinase activity that is present in microtubule preparations. We have now found that the activity copurifies with the projection portion of MAP 2 itself. Kinase activity coeluted with MAP 2 when microtubule protein was subjected to either gel- filtration chromatography on bio-gel A-15m or ion-exchange chromatography on DEAE- Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface. The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on bio-gel A-15m. Three protein species (M(r) = 39,000, 55,000, and 70,000) cofractionated with MAP 2, and two of these (M(r) = 39,000 and 55,000) may represent the subunits of an associated cyclic AMP- dependent protein kinase. The projection-associated activity was stimulated 10-fold by cyclic AMP and was inhibited more than 95 percent by the cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle. It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAPs, or the assembly-promoting domain of MAP 2, and was estimated to account for 7-22 percent of the total brain cytosolic protein kinase activity. The location of the kinase on the projection is consistent with a role in regulating the function of the projection, though other roles for the enzyme are also possible.     

Regulated association of microtubule-associated protein 2 (MAP2) with Src and Grb2: evidence for MAP2 as a scaffolding protein

Microtubule-associated protein 2 (MAP2) and tau, which is involved in Alzheimer's disease, are major cytoskeletal proteins in neurons. These proteins are involved in microtubule assembly and stability. To further characterize MAP2, we took a strategy of identifying potential MAP2 binding partners. The low molecular weight MAP2c protein has 11 PXXP motifs that are conserved across species, and these PXXP motifs could be potential ligands for Src homology 3 (SH3) domains. We tested for MAP2 interaction with SH3 domain-containing proteins. All neuronal MAP2 isoforms bound specifically to the SH3 domains of c-Src and Grb2 in an in vitro glutathione S-transferase-SH3 pull-down assay. Interactions between endogenous proteins were confirmed by co-immunoprecipitation using brain lysate. All three proteins were also found co-expressed in neuronal cell bodies and dendrites. Surprisingly, the SH3 domain-binding site was mapped to the microtubule-binding domain that contains no PXXP motif. Src bound primarily the soluble, non-microtubule-associated MAP2c in vitro. This specific MAP2/SH3 domain interaction was inhibited by phosphorylation of MAP2c by the mitogen-activated protein kinase extracellular signal-regulated kinase 2 but not by protein kinase A. This phosphorylation-regulated association of MAP2 with proteins of intracellular signal transduction pathways suggests a possible link between cellular signaling and neuronal cytoskeleton, with MAP2 perhaps acting as a molecular scaffold upon which cytoskeleton-modifying proteins assemble and dissociate in response to neuronal activity.     

Stoichiometry of estramustine phosphate binding to MAP2 measured by the disassembly of chick brain MAP2:tubulin microtubules

The concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5-6 moles.mole-1 estramustine phosphate, and that the Kd of these sites is congruent to 20 microM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin-binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.     

Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2)

This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.     

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.     

Domain structure and antiparallel dimers of microtubule-associated protein 2 (MAP2).

We have studied the microtubule-associated protein MAP2 from porcine brain and its subfragments by limited proteolysis, antibody labeling, and electron microscopy. Two major chymotryptic fragments start at lys 1528 and arg 1664, generating microtubule-binding fragments of Mr 36 kDa (303 residues, analogous to the "assembly domain" of Vallee, 1980) and 18 kDa (167 residues). These fragments can be labeled with the antibody 2-4 which recognizes the last internal repeat of MAP2 (Dingus et al., 1991). The epitope of another monoclonal antibody, AP18 (Binder et al., 1986), was mapped to the first 151 residues of MAP2. The interaction with AP18 is phosphorylation dependent; dephosphorylated MAP2 is not recognized. Intact MAP2 forms rod-like particles of 97 nm mean length, similar to Gottlieb and Murphy's (1985) observations. Both antibodies bind near an end of the rod, suggesting that the sequence and the structure are approximately colinear. There is a pronounced tendency for MAP2 to form dimers whose components are nearly in register but of opposite polarity. MAP2 can also fold in a hairpin-like fashion, generating 50-nm rods, and it can self-associate into oligomers and fibers. The 36-kDa microtubule-binding fragment also has a rod-like shape; its mean length is 49 nm, half of the intact molecule, even though the fragment contains only one-sixth of the mass. The antibody 2-4 decorates one end of the rod, similar to the intact protein. The fragment also forms antiparallel dimers, but its tendency for higher self-assembly forms is much lower than with intact MAP2.     

Chicken microtubule-associated protein 4 (MAP4): a novel member of the MAP4 family

 Chicken gizzard smooth muscle has often been used as a source of proteins of the contractile and cytoskeletal apparatus. In the present study, we isolated a hitherto unknown doublet of proteins, with apparent molecular weights of 200 kDa, from embryonic chicken gizzard and showed its association with the microtubular cytoskeleton by cosedimentation with microtubules (MTs) and by immunofluorescence staining of cultured cells. Immunoblot analysis also revealed the ubiquitous expression of this protein in all embryonic chicken tissues examined. Molecular cloning techniques allowed its identification as the chicken homologue of the microtubule-associated protein 4 (MAP4), known from mammalian species, and revealed approximately 90% of its amino acid sequence. MAP4 is the major MAP of non-neuronal tissues and cross-species comparisons clearly demonstrated its highly conserved overall structure, consisting of a basic C-terminal MT-binding region and an acidic N-terminal projection domain of unknown function. Despite these conserved features, overall sequence homologies to its mammalian counterparts are rather low and focused to distinct regions of the molecule. Among these are a conserved 18-amino acid motif, which is known to mediate binding to MTs and a part of the MT-binding domain known as the proline-rich region, which is thought to be the regulatory domain of MAP4. The N-terminal 59 amino acids are a conserved and unique feature of the MAP4 sequence and might be an indication that MAP4 performs other functions besides the enhancement of MT assembly. Accepted: 13 March 1996     

Differential binding regulation of microtubule-associated proteins MAP1A, MAP1B, and MAP2 by tubulin polyglutamylation

The major neuronal post-translational modification of tubulin, polyglutamylation, can act as a molecular potentiometer to modulate microtubule-associated proteins (MAPs) binding as a function of the polyglutamyl chain length. The relative affinity of Tau, MAP2, and kinesin has been shown to be optimal for tubulin modified by approximately 3 glutamyl units. Using blot overlay assays, we have tested the ability of polyglutamylation to modulate the interaction of two other structural MAPs, MAP1A and MAP1B, with tubulin. MAP1A and MAP2 display distinct behavior in terms of tubulin binding; they do not compete with each other, even when the polyglutamyl chains of tubulin are removed, indicating that they have distinct binding sites on tubulin. Binding of MAP1A and MAP1B to tubulin is also controlled by polyglutamylation and, although the modulation of MAP1B binding resembles that of MAP2, we found that polyglutamylation can exert a different mode of regulation toward MAP1A. Interestingly, although the affinity of the other MAPs tested so far decreases sharply for tubulins carrying long polyglutamyl chains, the affinity of MAP1A for these tubulins is maintained at a significant level. This differential regulation exerted by polyglutamylation toward different MAPs might facilitate their selective recruitment into distinct microtubule populations, hence modulating their functional properties.     

Phosphorylation determines the binding of microtubule-associated protein 2 (MAP2) to microtubules in living cells

The influence of phosphorylation on the binding of microtubule-associated protein 2 (MAP2) to cellular microtubules was studied by microinjecting MAP2 in various phosphorylation states into rat-1 fibroblasts, which lack endogenous MAP2. Conventionally prepared brain MAP2, containing 10 mol of endogenous phosphate per mol (MAP2-P10), was completely bound to cellular microtubules within 2-3 min after injection. MAP2 prepared in the presence of phosphatase inhibitors, containing 25 mol/mol of phosphate (MAP2-P25), also bound completely. However, MAP2 whose phosphate content had been reduced to 2 mol phosphate per mol by treatment with alkaline phosphatase in vitro (MAP2-P2) did not initially bind to microtubules, suggesting that phosphorylation of certain sites in MAP2 is essential for binding to microtubules. MAP2-P10 was further phosphorylated in vitro via an endogenously bound protein kinase activity, adding 12 more phosphates, giving a total of 22 mol/mol. This preparation (MAP2-P10+12) also did not bind to microtubules. Assay of the binding of these preparations to taxol-stabilized tubulin polymers in vitro confirmed that their binding to tubulin depended on the state of phosphorylation, but the results obtained in microinjection experiments differed in some cases from in vitro binding. The results suggest that the site of phosphate incorporation rather than the amount is the critical factor in determining microtubule binding activity of MAP2. Furthermore, the interaction of MAP2 with cellular microtubules may be influenced by additional factors that are not evident in vitro.     

Extensive cAMP-dependent and cAMP-independent phosphorylation of microtubule-associated protein 2

Microtubule-associated protein 2 (MAP 2) is the major substrate for phosphorylation in purified preparations of brain microtubules. In earlier work, we showed that phosphorylation is catalyzed by a type II cAMP-dependent protein kinase tightly associated with MAP 2 itself. In the present study, we have examined the extent of MAP 2 phosphorylation by its associated protein kinase. Using an inorganic phosphate assay, we found that MAP 2 contained from 8 to 13 mol of phosphate/mol of protein as isolated. The catalytic subunit of the MAP 2-associated kinase catalyzed the incorporation of additional phosphate to a final level of 20-22 mol/mol of MAP 2. Potato acid phosphatase was used to remove phosphate from MAP 2. Rephosphorylation of acid phosphatase-treated MAP 2 resulted in maximal incorporation of 13 mol of phosphate/mol of MAP 2. The rates and extent of [32P] phosphate incorporation into as isolated and dephosphorylated MAP 2 were found to be identical, and phosphate was incorporated into identical peptides in the two preparations. These results were interpreted to indicate that MAP 2 contains as many as 13 cAMP-dependent phosphorylation sites, and approximately eight phosphates of as yet undetermined origin.     

Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c).

Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.     

Cyclic nucleotide- and Ca2+-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1

MAP-2 and tubulin are both shown to be substrates for glycogen synthase (casein) kinase-1 (CK-1). Greater than 40 mol 32P is incorporated into MAP-2 by CK-1 compared to only 14 mol 32P observed when cyclic AMP-dependent protein kinase (A-kinase) is the catalyst. Peptide mapping shows that CK-1 and A-kinase recognize a few common sites; the majority of the sites phosphorylated on MAP-2 by CK-1 are quite distinct. Up to 4 mol 32P can be incorporated into the tubulin dimer by CK-1 compared to only 0.9 mol 32P by A-kinase. The preferred substrate for both kinases is beta-tubulin.     

Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.     

Comparative measurement by radioimmunoassay of the brain microtubule-associated protein MAP2

Summary The presence of the microtubule-associated protein (MAP₂) in the brain of several species has been investigated by SDS-gel electrophoresis and by radioimmunoassay. This assay had a sensitivity of approx. 10 ng and it was capable of measuring the protein either in purified microtubules or in crude brain extracts. As determined with this radioimmunoassay, MAP₂ accounted for about 10% of the porcine brain microtubule protein and 1% of the protein from a brain extract. Taking porcine MAP₂ as a reference, we have detected polypeptides with the same electrophoretic mobility in brain microtubules from cow, sheep, rat and chicken. Nevertheless, the MAP₂ from these species showed a variable degree of immunoreactivity. Bovine MAP₂ appeared closely related to the porcine protein whereas the rat antigen showed low cross-reaction and chicken MAP₂ appeared immunologically unrelated to porcine MAP₂. Our results suggest a higher variability of the MAP₂ sequences as compared to that reported by other authors for the brain microtubule protein, tubulin.     

Involvement of microtubule-associated protein 2 (MAP2) in oral cancer cell motility: a novel biological function of MAP2 in non-neuronal cells

Microtubule-associated protein 2 (MAP2) has been better known for its well-defined role primarily in neurite outgrowth during neuronal development. However, the biological functions of MAP2 in non-neuronal cells, such as epithelial cells, remain largely unknown. In the present study, we sought to investigate the cellular functions of MAP2 by separately establishing stable expression of two MAP2 isoforms, MAP2A and MAP2C, in oral squamous cell carcinoma, Ca9-22. Ectopic expression of MAP2A or MAP2C results in microtubule bundling predominantly at the cell periphery. Remarkably, overexpression of MAP2A but not MAP2C significantly promotes migration of Ca9-22 cells, whereas knockdown of MAP2A expression by specific siRNA oligos dramatically decreases cell migration of HaCaT, an immortalized keratinocyte cell line with abundant endogenous MAP2A. Furthermore, by immunohistochemical studies, MAP2A was shown to highly and selectively express in invasive oral cancer tissues, consistent with its motility-promoting cellular function revealed through in vitro assays. Thus, our findings have not only identified a novel role of MAP2 in non-neuronal cells, but also provided the first implication of MAP2 in malignant oral cancer tissues.