Proc. Natl. Acad. Sci. USAVol. 77, No. 6, pp. 3206-3210, June 1980Biochemistry

Structure and phosphorylation of microtubule-associatedprotein 2 (MAP 2)

(microtubule assembly/cyclic AMP-dependent protein kinase/microtubule structure)

RICHARD VALLEECell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

Communicated by Paul Greengard, March 3, 1980

ABSTRACT Chymotryptic fragments of microtubule-as-sociated protein 2 (MAP 2) containing the portion of the mole-cule responsible for promoting microtubule assembly wereidentified. These assembly-promoting fragments displaced in-tact MAP 2, but not MAP 1, from assembled microtubules. Thisindicates that the association of MAP 2 with the microtubulesurface is reversible. Both the assembly- romoting fragmentsand fragments representing the portion of the MAP 2 moleculeobserved as a projection on the microtubule surface were foundto contain sites for endogenous cyclic AMP-dependent phos-phorylation. The projection fragments were capable of endog-enous phosphorylation even after their physical separation frommicrotubules. This suggests an intimate association of a kinaseactivity with the projections. Detailed analysis of the propertiesof the chymotryptic fragments of MAP 2 has led to a map of themolecule showing the major sites of proteolytic attack and thesites of phosphorylation.

A number of proteins copurify with brain microtubules and onthe basis of this behavior have been termed microtubule-asso-ciated proteins or MAPs. Most prominent among these arespecies of high subunit molecular weight that have been re-ferred to collectively as HMW (1) or individually as MAP 1 andMAP 2 (2). While MAP 1 has not been well characterized, MAP2 has been the subject of several recent studies. It appears as aprojection on the microtubule surface and also has the propertyof promoting microtubule assembly in vitro (3-8). Thus, MAP2 may serve two functions in the cell: mediating the interactionof microtubules with other cellular structures and controllingmicrotubule assembly.We have suggested that these two functions may be associ-

ated with discrete parts of the MAP 2 molecule (5). In ourearlier study involving the controlled digestion of microtubuleswith trypsin, a large fragment of MAP 2 that had lost the abilityto bind to microtubules was identified. This fragment corre-sponded to the projection observed on the microtubule surfaceby electron microscopy (5). In the present study, using chy-motrypsin as the protease, a second portion of the molecule notdetected by electron microscopy has been identified; it retainsthe ability to bind to microtubules and is responsible for con-trolling microtubule assembly. This portion has been charac-terized and its properties as well as a number of properties ofthe projection are described in this report.

MATERIALS AND METHODSProcedures for Preparing Protein. Microtubule protein was

purified from calf cerebral cortex by the reversible assemblymethod of Borisy et al. (1), using an assembly buffer consistingof 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), pH1 6.6at 250C, 0.1 mM EDTA, 1.0 mM ethylene glycol bis(f3-ami-

noethyl ether)-NN,N',N'-tetraacetic acid, 1.0mM MgSO4, and1.0 mM GTP, plus 1.0 mM 2-mercaptoethanol at the extractionstage. Tubulin was purified by ion-exchange chromatographyon DEAE-Sephadex (9) at pH 6.6. A crude MAP fraction wasprepared by exposure of microtubules to elevated temperatures(10). MAP 2 was further purified by gel filtration chromatog-raphy on a 1.5 X 25 cm column of Bio-Gel A-15m equilibratedwith assembly buffer, to remove the T MAPs (11) present in thecrude MAP fraction (7, 8).

Digestion of MAP 2. Digestion with a-chymotrypsin(Worthington) was performed in assembly buffer at 370C (seefigure legends for other reaction conditions). The reaction wasquenched with 2 mM phenylmethylsulfonyl fluoride, a leveldetermined to block completely the digestion of MAP 2 for atleast 1 hr at 37°C.

Phosphorylation of MAP 2. Endogenous phosphorylationwas carried out under conditions based on those of Sloboda etal. (2). The reaction mixture contained microtubules at 5mg/ml in assembly buffer, 5-10 mM MgSO4, I 10 ,uM cyclicAMP, and 25 ,uM ATP, including approximately 20,uCi/ mlof ['y-32P]ATP at 20-40 Ci/mmol (1 Ci = 3.7 X 1010 becquer-els). Samples were preincubated at 37°C for 2 min and the re-action was initiated by addition of the ATP. The reaction wasterminated by addition of electrophoresis sample buffer(12).

Analytical Methods. Electrophoresis was conducted in1.5-mm-thick slab gels, with 9% acrylamide in the running gel(12). Samples were incubated in a boiling water bath for 1 minimmediately after mixing with sample buffer. Gels were stainedwith Coomassie brilliant blue R 250 (CBB) (13) and quanti-tated by densitometry in what was determined to be the linearrange of absorbance as a function of protein concentration (14).Molecular weight standards were MAP 2, skeletal muscle my-osin, a-actinin, bovine serum albumin, actin, and a-chymo-trypsinogen. Protein concentration was determined (15, 16),using bovine serum albumin as a standard. Autoradiographywas performed by using Kodak X-Omat R film without an in-tensifying screen. To determine the amount of phosphate in-corporated into protein, the MAP 2 or fragment bands wereexcised from stained and dried gels. 32P radioactivity was thendetermined by liquid scintillation counting in Aquasol (NewEngland Nuclear).

RESULTSMicrotubule-binding and assembly-promoting activityof MAP 2 fragmentsIn preliminary experiments it was found that, as had been ob-served with trypsin (5), chymotrypsin removed the projectionsfrom the surface of microtubules. Microtubule assembly was

Abbreviations: MAP, microtubule-associated protein; HMW, highmolecular weight protein; CBB, Coomassie brilliant blue R 250.

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FIG. 1. Microtubule-binding and nonbinding fragments of MAP 2. (A) Electrophoretic gel showing purified MAP 2. (B) Electrophoreticgel showing cosedimentation of MAP 2 fragments with microtubules. Purified MAP 2 at 2.1 mg/ml was exposed to chymotrypsin at 0.7 jig/mlfor 0, 2, and 8 min. Pure tubulin was combined with the three samples (lanes labeled 0', 2', and 8') in assembly buffer, or with buffer alone (laneslabeled T). Final concentrations were 1.4 mg/ml for tubulin and 0.6 mg/ml for MAP 2 or fragments. The mixtures (lanes labeled Total) wereincubated at 370C for 10 min to allow microtubules to assemble. They were then centrifuged at 30,000 X g for 20 min at 370C, yielding a supernatantfraction (lanes labeled Cycle I, Super) containing nonbinding fragments of MAP 2, and a pellet containing microtubules. Fractions referredto as Pellet were prepared by resuspension of the pellets, depolymerization of the microtubules at 0C, and centrifugation at 30,000 X g for 20min at 40C (lanes labeled Cycle I, Pellet). These fractions contained fragments ofMAP 2 that had bound to microtubules at 370C and disassembledwith microtubules at 0C. These samples were reassembled, and supernatant and pellet fractions were again recovered (Cycle II). Molecularweights are in thousands. Tub, tubulin.

almost unaffected by this treatment (sedimentable microtubulesdeclining at only 1.1%/min, compared to a 90% loss of intactMAP 2 by 2 min and complete loss by 8 min of digestion). Thisagain suggested that, as with trypsin, a discrete assembly-pro-moting portion of the MAP 2 molecule remained bound to themicrotubules. This portion of MAP 2 was first identified byusing chymotrypsin; this protease was, therefore, used in thefurther studies described in this report. It was also determinedthat identical chymotryptic fragments were produced withmicrotubules (see Fig. 3), heat-treated MAPs (Fig. 2), or puri-fied MAP 2 (Fig. 1) as substrate. This permitted the followingexperiment to be performed, in which purified MAP 2 was usedas substrate to identify the assembly-promoting portion of MAP2.

Purified MAP 2 (Fig. 1A) was exposed to chymotrypsin fora series of times (0, 2, and 8 min, Fig. 1B). The samples were

then combined with tubulin. The mixtures (Total, 0', 2', and8' samples), as well as tubulin alone (Total, sample T) were in-cubated at 37'C to allow microtubules to assemble and were

then centrifuged. The supernatants (Cycle I Super samples)contained components that were not incorporated into mi-crotubules. Microtubule pellet fractions (Pellet) containedproteins or fragments incorporated reversibly into microtubules.The assembly/disassembly cycle was repeated a second time(Cycle II), yielding comparable Super and Pellet fractions.

Tubulin alone (Total, sample T) failed to assemble signifi-cantly, most of the protein remaining in the supernatant (CycleI, samples T, Super vs. Pellet). Addition of undigested MAP 2(Total, 0' sample) resulted in extensive microtubule assembly,with most of the tubulin and MAP 2 found in the microtubulepellet (Cycles I and II, 0' Pellet samples). Exposure of MAP 2to chymotrypsin resulted in extensive reduction of the MAP 2

band by 2 min and its complete disappearance by 8 min (Total,2' and 8' samples). Microtubule assembly was only slightlydecreased, as indicated by the excess of tubulin in the Pellet vs.the Super fractions at 2 and 8 min of digestion.The fragments of MAP 2 (Mr 270,000)* produced by chy-

motrypsin differed in their abilities to bind to microtubules. Afragment of Mr 240,000 appeared early in the reaction (Total,2' sample) and did not sediment with microtubules (Cycle I,Super, 2' sample). This species was identified as the projectionportion of MAP 2. It was indistinguishable in size from thecomparable fragment produced by trypsin (5) even on 4%acrylamide gels, indicating that the sites of cleavage by trypsinand chymotrypsin are quite close to each other in the MAP 2polypeptide chain. Other prominent fragments of Mr 180,000and 140,000 were also observed in the supernatant (Cycle I,

Super, 2' and 8' samples) and are probably subfragments of theprojection.

Fragments of Mr 215,000, 185,000, and 32,000-39,000 co-

sedimented with microtubules after 2 min of digestion (CycleI, Pellet, 2' sample). By 8 min only a pair of fragments of Mr32,000 and 34,000 remained from this group (Cycle I, Pellet,8' sample). The fragments of Mr 32,000-39,000 were in therange of size expected for the assembly-promoting portion ofMAP 2 (Mr 270,000 for MAP 2- 240,000 for the projection =30,000). They arose with a time course similar to that for thedisappearance of intact MAP 2 and to a molar level close to theinitial level of MAP 2 (data not shown). In addition to binding

* MAP 2 from pig (HMW B in ref. 5) and from calf brain consists oftwo species of Mr 286,000 and 271,000 (which is rounded to 270,000in this paper), the former being the more prominent in pig, the latterin calf. The projection portion of MAP 2 appears as a doublet on gelswith a major band at Mr 255,000 in pig (5) and 240,000 in calf.

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to microtubules, fragments from the Mr 32,000-39,000 grouppromoted the assembly of pure tubulin into microtubules(compare Pellet and Super, 8' samples, in both Cycle I and II).In view of these properties, it was concluded that the Mr32,000-39,000 fragments represented the portion of MAP 2 thatis responsible for binding to microtubules and for promotingmicrotubule assembly.

Do the fragments occupy the same sites on themicrotubules as does intact MAP 2?To test whether MAP 2 and what will now be referred to as theassembly-promoting fragments (Mr 32,000-39,000) occupiedthe same sites on the microtubule surface, the following ex-

periment was performed (Fig. 2). Microtubules were assembledfrom a crude MAP fraction (10) plus tubulin (Fig. 2A, lane a).The microtubules were layered over a digest of the same MAPfraction in 20% sucrose (lane b) and then sedimented. Themicrotubule pellet is shown in lane c. MAP 2 was almost totallyreplaced (90% reduction) by fragments of Mr 32,000-39,000when microtubules were sedimented through the digest. Thedisplaced MAP 2 was recovered intact in the supernatant (notshown). The level of MAP 2 was not reduced when microtu-bules were sedimented through sucrose alone (lane d). Identicalresults were obtained when 0.1 mM colchicine was includedwith the digest. This concentration of colchicine totally blockednew microtubule assembly but had no detectable effect on theamount of preformed microtubules sedimented or on the dis-placement of MAP 2 by its fragments. Together these resultsconstitute evidence for the reversible association of a MAP withthe microtubule surface. It was also found that MAP 2 displaced

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FIG. 2. Electrophoretic gel showing displacement of MAP 2 byfragments. Fragments were prepared by exposure of a crude MAPfraction (10) at 4.0 mg/ml to chymotrypsin at 1.3 ,ug/ml for 8 min. (A)Microtubules (lane a) were prepared in assembly buffer by mixingcrude MAPs (final concentration 1.6 mg/ml) with tubulin (finalconcentration 2.8 mg/ml). The microtubules were layered over 5 volof the crude MAP digest (lane b) at 2.0 mg/ml in assembly buffercontaining 20% sucrose. The MAP 2 fragments were present in a

6.25-fold molar excess in the sucrose layer vs. the overlayered mi-crotubules. The microtubules were sedimented through the fragmentsat 50,000 X g in a swinging bucket rotor for 45 min at 30'C. Mi-crotubules sedimented through sucrose alone in assembly buffer are

shown in lane d. (B) Same as A, except that four-cycle purified (1)whole microtubule protein (lane a) at 5 mg/ml was layered over thecrude MAP digest.

the fragments from microtubules (data not shown). Thus, thefragments appear to occupy the same sites on the microtubulesurface as does the intact MAP 2 molecule.

In the experiment shown in Fig. 2B, unfractionated mi-crotubules containing MAP 1 as well as MAP 2 (lane a) weresedimented through the MAP digest (lane b). MAP 2 was re-duced by 90% while MAP 1 was almost unchanged (lane c)compared to microtubules sedimented through sucrose alone(latie d). This result indicates that the regions of MAP 1 andMAP 2 that are responsible for microtubule binding may insome way differ, either in structure or in chemical composi-tion.Phosphorylation of the assembly and projectionportions of MAP 2Microtubule preparations contain a protein kinase activity thatquantitatively copurifies with microtubules and that is re-sponsible for the specific phosphorylation of MAP 2 (2, 17). Thisreaction could modulate either the assembly-promoting activityof MAP 2 or the interaction of the projection with cellular or-ganelles. The following experiments were performed to de-termine which portion of the MAP 2 molecule is phosphoryl-ated and, hence, the likely role of the phosphorylation reac-tion.

As for microtubules purified from chicken and rat brain (2,17), it was found that MAP 2 was the predominant speciesphosphorylated in microtubules purified from calf brain (Fig.3A). Phosphate incorporation was 2.15 + 0.20 mol per mol ofcalf MAP 2 in vitro, close to the previously reported value of1.9 mol/mol for chicken MAP 2 (2). To examine the phos-phorylation of the MAP 2 fragments, microtubules werephosphorylated either before or after exposure to chymotrypsin.The pattern of phosphorylation was unaffected by the orderin which these steps were performed. Another variation on theseprocedures was used to identify phosphorylated fragments onthe basis of their ability to bind to microtubules and is shownin Fig. 3B. Microtubules were first exposed to chymotrypsinand then sedimented to separate microtubule-binding fromnonbinding fragments of MAP 2. The microtubule pellets wereresuspended and the pellet and supernatant fractions were in-dependently phosphorylated. Fig. SB shows a gel of the pelletand supernatant samples for a series of times of chymotrypsindigestion, stained with CBB to reveal total protein. Also shownis an autoradiograph of the same gel, revealing phosphorylatedprotein species.

It may be observed that both microtubule-binding (32P, Pelletsamples) and nonbinding (32p, Super samples) fragments ofMAP 2 were phosphorylated. Thus, phosphorylation sites arelocated on both the projection and assembly-promoting portionsof MAP 2. A number of fragments identified in Figs. 1 and 2were phosphorylated. These included the assembly-promotingfragments (Mr 32,000-39,000) as well as microtubule-bindingspecies of Mr 215,000 and 185,000 (compare 32p with CBBPellet samples). The projection fragment (Mr 240,000), anonbinding species of 180,000, and a species not earlier iden-tified and not detected by CBB stain (Mr 35,000) were alsoprominently phosphorylated (compare 32p with CBB Supersamples). Notable in its lack of prominence in the autoradio-graph was a nonbinding species at Mr 140,000 (Fig. 1; Fig. 3B,CBB vs. 32p Super samples). This species has shown a variablelow level of phosphorylation that, in contrast to the otherfragments (see below), was not stimulated by cyclic AMP.

Table 1 shows the effect of cyclic AMP on the phosphoryl-ation of MAP 2 and its fragments. Phosphorylation of MAP 2was stimulated 2-fold by cyclic AMP. Phosphorylation of theprojection and the assembly-promoting portions of MAP 2 wasstimulated by cyclic AMP to a similar extent.

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1 2 3 4 1 2 3 4 D 1 2 3 4 5 1 2 3 4 nFIG. 3. Electrophoretic gels showing phosphorylation of intact or chymotrypsin-digested MAP 2. Microtubules were prepared by two cycles

of assembly/disassembly purification (1). Endogenous phosphorylation took place in the presence of cyclic AMP for 2 min at 370C. Sampleswere analyzed by electrophoresis. The electrophoretic gels were stained with Coomassie brilliant blue (CBB) and were also autoradiographed(32p). (A) Intact microtubules. (B) Microtubules exposed to chymotrypsin. Assembled microtubules at 5 mg/ml were exposed to chymotrypsinat 0.35 jig/ml for a series of times at 370C, aliquots were removed, and the reaction was quenched with 2 mM phenylmethylsulfonyl fluoride.The microtubules were sedimented at 30,000 X g for 30 min. The pellets were resuspended to the initial volume and contained tubulin (Tub)and the microtubule-binding fragments of MAP 2. The supernatants (Super) contained MAP 2 fragments not bound to microtubules, and asmall amount of tubulin. Phosphorylation was conducted for 2 min in the presence of cyclicAMP as for intact microtubules, but using the separatedfractions as substrate. Times of exposure to chymotrypsin for lanes 1-5 were 1, 2, 4, 8, and 16 min, respectively.

It should be noted that in the experiment shown in Fig. 3 theprojection fragments were phosphorylated despite their priorseparation from microtubules. This observation, along with thecopurification of the kinase activity with microtubules (2),suggests that a "projection kinase" could be specifically asso-

ciated with the projection. Whether a MAP 2 "assembly kinase"is bound to the assembly-promoting portion of MAP 2 or tosome other portion of the microtubule is a subject for furtherinvestigation.

Table 1. Dependence of phosphorylation on cyclic AMPPhosphorylation, Fold

cpm/mol stimulationElectrophoretic - cyclic + cyclic by cyclic

species AMP AMP AMP

MAP 2 2460 + 180 4980 I 700 2.0240,000 (projection

fragment) 1307 + 104 2890 ± 470 2.232,000-39,000 (assembly

fragments) 500 ± 36 1180 ± 24 2.4

Three-cycle microtubules (1) were phosphorylated in the presenceor absence of cyclic AMP for 2 min. The reactions were quenched byincubation in a boiling water bath after addition of 0.75M NaCi and10 mM dithiothreitol, conditions used as a first step in preparing acrude MAP fraction (10). After centrifugation to remove coagulatedtubulin and dialysis against assembly buffer, the two MAP prepara-tions (with or without cyclic AMP) were exposed to chymotrypsin fora series of times. As described for Fig. 1, tubulin was added to thefractions, microtubules were assembled, and microtubule-bindingand nonbinding fragments ofMAP 2 were separated. 32p radioactivityand CBB stain were determined for two or three replicate samplesfor each electrophoretic species. CBB stain was determined in arbi-trary units; hence cpm/mol = cpm/(bound stain/molecular weight)is also in arbitrary units. Values are given as mean + SD.

DISCUSSIONI have shown that both trypsin (5) and chymotrypsin can beused to divide the MAP 2 molecule into projection and assem-

bly-promoting portions. In the present experiments a group ofchymotryptic fragments of MAP 2 of Mr 32,000-39,000 havebeen identified as deriving from the assembly-promoting andmicrotubule-binding portion of MAP 2. The basis for thisidentification is as follows. (i) The fragments were producedfrom highly purified MAP 2 (Fig. 1); (ii) they were producedat a molar level comparable to the initial level of intact MAP2 and with a time course closely corresponding to the disap-pearance of intact MAP 2 (see Fig. 1; full data not shown); (iii)they were of the expected size (5) for the assembly-promotingportion of MAP 2; (iv) they bound strongly to microtubules(Figs. 1-3); (v) they promoted microtubule assembly (Fig. 1);and (vi) they displaced intact MAP 2 from the microtubulesurface (Fig. 2), indicating that they occupied the same sites asthe intact MAP 2 molecule.

Fragment map of MAP 2

The primary site of tryptic or chymotryptic cleavage of theMAP 2 molecule appears to lie between the assembly-pro-moting and projection portions of the molecule. However, thedigestion process is not entirely restricted to this site. Additionalfragments of MAP 2 produced after the initial cleavage intoassembly and projection portions may be accounted for by twoadditional sites of proteolytic attack, as shown in Fig. 4. It isassumed that in the elongated MAP 2 molecule proteolytic at-tack at each site is independent of attack at the other sites. Thedigestion process would proceed in parallel at the three sites butat different rates, an assumption that has been used successfullyto describe the digestion of other proteins (18). The schemeoutlined in Fig. 4 predicts the existence of three fragments

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FIG. 4. Fragment map of MAP 2. The shaded structure repre-sents the extended MAP 2 polypeptide chain. The region of themolecule involved in microtubule binding is shown schematicallyassociated with a microtubule at the left. Proposed sites of cleavageby chymotrypsin along the primary sequence of MAP 2 are indicatedby arrows in order of decreasing rate of attack by the protease.Phosphorylation sites are indicated by asterisks. Molecular weightsare given in thousands.

containing the assembly-promoting region of MAP 2 and,therefore, capable of binding to microtubules. Cleavage at site1 would produce a fragment of 35,000 daltons, cleavage at site2 a fragment of 210,000 daltons, and cleavage at site 3 a frag-ment of 175,000 daltons. These would correspond to the32,000-39,000-dalton, 215,000-dalton, and 185,000-daltonmicrotubule-binding species observed in Figs. 1 and 3. Themodel also predicts the existence of nonbinding fragments ofMr 235,000, 175,000, 140,000, 95,000, 60,000, and 35,000.Nonbinding fragments with molecular weights quite close tomost of these values (Mr 240,000, 180,000, 140,000, and 35,000)are prominent in Figs. 1 and 3. A fragment of Mr 95,000 wouldnot be expected to be prominent because it would be formedat a relatively slow rate by cleavage at site 3, and destroyedrapidly by cleavage at site 2. Nonetheless, a fragment band ofabout this size has been observed on occasion (unpublishedresults) and could represent the species in question. The pre-dicted species of Mr 60,000 may correspond to a fragment ofMr 71,000 observed in Fig. 3. Why this band is not more

prominent is unclear. This segment of MAP 2, like the adjacent35,000-dalton segment (Fig. 3), may stain weakly with CBB,or it may be further digested at sites that become accessible tothe protease only after cleavage at site 2. Despite these minorpoints of uncertainty, the fragment map of MAP 2 shown inFig. 4 is closely consistent with the data and provides insightinto the substructure of the molecule.

Phosphorylation of MAP 2MAP 2 of chicken brain has been reported to accept approxi-mately 2 mol of phosphate per mol of protein as the result ofa protein kinase activity that copurifies with microtubules (2).In the present study a similar value was obtained for the numberof phosphates incorporated into calf MAP 2. Both microtub-ule-binding and nonbinding fragments were phosphorylated.This suggests that there could be one site of phosphorylation on

the assembly-promoting portion of MAP 2 and a second on theprojection, though it must be stressed that the data do not ex-

clude multiple sites on either part of the molecule. On the basis

of the state of phosphorylation of the chymotryptic fragmentsof MAP 2 (Fig. 3), the phosphorylation sites have been assignedto specific positions (asterisks) on the fragment map shown inFig. 4. One site or group of sites is assigned to the assembly-promoting portion of MAP 2. Another is assigned to the segmentof the molecule lying between cleavage sites 2 and 3 (Mr35,000). Under conditions in which no further phosphorylationis detected, nonbinding fragments of Mr 235,000, 175,000,95,000, and 35,000 and all microtubule-binding fragmentsshould be phosphorylated, while the fragments of Mr 140,000and 60,000 should not. This is close to what was actually ob-served (Fig. 3).

Phosphorylation of both portions of MAP 2 was stimulatedby cyclic AMP. Therefore, an effect of cyclic AMP-dependentphosphorylation on both the assembly-promoting activity ofMAP 2 and its interaction with other cellular structures is pos-sible. It will be of considerable interest to determine the natureof these effects as well as the positions of the phosphorylationsites in the folded MAP 2 molecule and the sites of binding ofthe kinases.

I thank Michael DiBartolomeis for doing much of the phosphoryl-ation work and Vikram Patel and Grant Fairbanks for valuable dis-cussions. This study was supported by National Institutes of HealthGrant P30 12708, Grant GM26701 to R.V., and the Mimi AaronGreenberg Fund.

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6. Murphy, D. B., Vallee, R. B. & Borisy, G. G. (1977) Biochemistry16,2598-2605.

7. Herzog, W. & Weber, K. (1978) Eur. J. Biochem. 92, 1-8.8. Kim, H., Binder, L. I. & Rosenbaum, J. L. (1979) J. Cell Biol. 80,

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