Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 177-181, January 1975

Cyclic AMP-Dependent Endogenous Phosphorylation of a -Associated Protein (protein phosphorylation/in vitro assembly) ROGER D. SLOBODA, STEPHEN A. RUDOLPH, JOEL L. ROSENBAUM, AND PAUL GREENGARD Department of Biology, Kline Biology Tower, Yale University and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06610 Communicated by Aaron B. Lerner, October 21, 1974

ABSTRACT prepared from chick brain were homogenized in a motor-driven glass-Teflon homogenizer homogenates by successive cycles of assembly-disassembly at 00 in 1 ml of polymerization mixture buffer [1 mM MgSO4, were found to contain two high-molecular-weight pro- ether)-N,N'-tetra- teins, designated microtubule-associated protein, and 2 mM ethyleneglycol bis(#-aminoethyl microtubule-associated proteins. Microtubule-associated acetate (EGTA), 1 mM GTP, 100 mM piperazine-N,N'-bis(2- protein2 (apparent molecular weight 300,000 by sodium ethanesulfonic acid) (Pipes) (pH 6.9)] per g wet weight, and dodecyl sulfate-polyacrylamide gel electrophoresis) was the homogenate was centrifuged at 130,000 X g for 1 hr at 40 the preferred substrate for an endogenous cyclic AMP- to yield a crude high-speed supernatant. This supernatant was dependent protein kinase which appeared to be an integral component of the microtubules. The initial rate of phos- diluted with an equal volume of polymerization mixture buffer phorylation of microtubule-associated protein2 was en- containing 8 M glycerol, and incubated at 370 for 30 min to hanced 4- to 6-fold by cyclic AMP, with half-maximal assemble microtubules. The microtubules were sedimented by stimulation occurring at 2 X 10-7 M cyclic AMP. Under centrifugation at 130,000 X g for 1 hr at 250, resuspended in optimal conditions, a total of 1.0 and 1.9 mol of phosphate the volume of was incorporated per mole of microtubule-associated polymerization mixture buffer at 00 (1/4-1/3 protein2, in the absence and presence of cyclic AMP, re- the high speed supernatant) with a Dounce homogenizer, and spectively. Cyclic AMP also stimulated the phosphoryla- incubated at 0° for 30 min to depolymerize the microtubules. tion of , but the rate of phosphate incorporation The solution was then centrifuged at 130,000 X g for 30 min at per mol of tubulin was only 0.15% that of microtubule- material not dissociated by the cold treat- associated protein2. The data raise the possibility that the 40 to sediment any cyclic AMP-dependent phosphorylation of microtubule- ment. The supernatant from this centrifugation was called associated protein2 may play a role in microtubule assem- 1 X-polymerized microtubule protein. The 1 X-polymerized bly or function. microtubule protein was further purified by diluting with an equal volume of polymerization mixture buffer plus glycerol It has been reported that cyclic AMP stimulates the phos- and repeating the above procedure; in these studies, up to five phorylation of tubulin, the major microtubule protein (1, 2). cycles of successive assembly-diassembly were carried out, to Moreover, microtubule preparations have been found to yield 2X- to 5X-polymerized microtubule protein. Occa- contain a cyclic AMP-dependent protein kinase that can sionally, the microtubule protein preparation was stored over- catalyze the phosphorylation of exogenous substrates (3-5). night in 8 M glycerol in polymerization mixture buffer at When microtubule protein is isolated by in vitro assembly- -200. disassembly methods (6, 7) and the proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Phosphorylation In Vitro. The standard reaction mixture two proteins of high molecular weight are found to co-purify contained 50 mM 2(N-morpholino)ethanesulfonic acid (Mes) with the tubulin (8-11). In this report, evidence is (pH 6.2), 10 mM MgSO4, 1-2 MM [y-32PJATP [specific presented showing that such microtubule preparations contain activity 10-20 Ci/mmol, prepared by the method of Post and a cyclic AMP-dependent protein kinase whose preferred sub- Sen (13)], and 50 .g of microtubule protein, with or without strate is the faster migrating of the two high molecular weight 10MAM cyclic AMP, in a volume of 100 Ml. The phosphorylation proteins that co-purify with the . This endog- reaction was initiated by the addition of the [y-82P]ATP. enous nontubulin substrate incorporates stoichiometric Incubations were carried out for 30 sec at 300 in a Dubnoff amounts of phosphate under the experimental conditions metabolic shaker. The reaction was terminated by the addi- studied. tion of 50 ul of a solution containing 9% sodium dodecyl sulfate, 30 mM Tris-acetate (pH 7.8), 3 mM EDTA, and 15% MATERIALS AND METHODS glycerol. The reaction tubes were then transferred to a boiling Preparation of Microtubules. Microtubule protein was puri- water bath for 2 min, after which 25 Ml of 0.6 M dithiothreitol fied from brain homogenates of 1- to 3-day-old chicks by a were added. Aliquots (75 Ml) were subjected to electrophoresis modification of the method of Shelanski et al. (12). Brains on 5.6% polyacrylamide slab gels containing 1% sodium dodecyl sulfate (14). The gels were stained with Coomassie Abbreviations: MAP, microtubule-associated protein; Pipes, blue and dried. Autoradiography was carried out as described piperazine-N,N'-bis(2-ethanesulfonic acid); Hepes, N-2-hydroxy- (15, 16). The amount of 82p incorporated into the individual ethylpiperazine-N'-2-ethanesulfonic acid; Mes, 2(N-morpholino)- protein bands was measured either by liquid scintillation ethanesulfonic acid. counting of gel slices (Figs. 3 and 4) or by scanning the auto- 177 Downloaded by guest on September 23, 2021 i 1-8 Biochemistry: Sloboda et al. Proc. Nat. Acad. Sci. USA 72 (1975)

Molec u lar A u t o - r o n W e i g h t P t e i radiograph S t a i n i n g M A _bNW--m SEX.. .- Pi

- gin--.- 000000 a M A P

-- 2 0 0,0 0 0 r -

- 1 50,0 0 0--

100,0 0 0

-T u b u I in Tubu in-i -4 - 5 55000 _u

+ - cA MP

FIG. 1. Effect of cyclic AM\IP on the endogenous phosphorylation of microtubule proteins. Fifty micrograms of 3 X>-polymerized micro- tubule protein were phosphorylated for 30 sec, in the standard reaction mixture, in the presence or absence of 10-5 MI cyclic AMP. An aliquot containing 25 ,g of the sodium dodecyl sulfate-solubilized protein was then subjected to polyacrylamide gel electrophoresis. The autoradiograph, showing 32p incorporation, is on the left, and the protein staining pattern is on the right. MAP, microtubule-associated protein.

radiographs with a Canalco model G-I microdensitometer transport, and the areas under the peaks were determined (Fig. 1, Tables 1 and 2). with a planimeter. Total protein in microtubule preparations was determined by the Schacterle and Pollack modification Protein Measurement. In order to determine the relative (19) of the procedure of Lowry et al. (20). amounts of individual proteins at each stage of the purifica- tion, samples were separated by sodium dodecyl sulfate- Labeling In Vivo. A mixture of 5 mCi of [32P]orthophosphate polyacrylamide gel electrophoresis by the method of Laemmli (New England Nuclear Corp., carrier-free as phosphoric acid) (17). This procedure, unlike the method of Fairbanks et al. and 2.5 mCi of [3H]leucine (New England Nuclear Corp., (14), resolves tubulins 1 and 2. The proteins were fixed and the L- [4,5-3H ]leucine, 30 Ci/mmol) was evaporated to dryness and sodium (lodecyl sulfate was eluted by a 24-hr rinse in 50% resuspended in 35 ,l of phosphate-buffered saline (0.15 M methanol-7% acetic acid (v/v); the proteins were then quan- titatively stained in 1% Fast Green (18) in 50% methanol-7% TABLE 2. Relative amounts of major proteins and initial rate acetic acid (v/v) for 18 hr. The gels were destained for 24 hr in of J1AP2 phosphorylation in microtubule preparations subjected 5% methanol-7% acetic acid (v/v) in a Hoeffer diffusion to repeated cycles of assembly-disassembly destainer. The destained gels were scanned at 650 nm in a Gilford model 240 spectrophotometer equipped with linear Initial rate of MAP2 TABLE 1. Phosphorylation of protein components Tubulin Tubulin phosphoryl- of microtubbules Preparation 1* 2* MAP,* MlAP2* ationt

M\1ol of Initial rate of High-speed phosphorylation supernatant 18.0 18.8 0.2 0.5 <0. 1 protein Percent 2.2 2.8 3.5 of total (mol of phosphate/mol 1X 33.6 38.8 component 2X 36.4 41.9 2.2 2.9 4.5 Protein per 100 mol incorporated of protein per 37.1 39.9 2.3 3.1 5.1 of tubulin phosphate min) X 10l 3X component 4X 36.6 40.5 2.5 3.1 5.0 MAP, 0.41 0 0.0 5X 39.0 42.6 2.4 3.3 5.0 MAP2 0.69 65 90.0 Tubulin 100.0 15 0.14 Protein was measured on polyacrylamide gels stained with Fast Green as described in Materials and Methods. 3X-Polymerized microtubule protein was phosphorylated for * Expressed as weight percent of total protein. 30 see in the standard reaction mixture in the presence of 10-5 MI t For each preparation, the initial rate of phosphorylation, cyclic AMIP. Measurements of protein and of phosphate in- in the presence of cyclic AMXP, is expressed as pmol of phosphate corporation were performed as described in Materials and incorporated into MIAP2 per gel slot (containing 25 Ag of total Methods. protein) per min. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 72 (1976) Phosphorylation of Microtubule-Associated Protein 179

61 .20 _ 84F C .18 tubulin ~.E 2 .162 56 e 1 R~~ A6 50 i2 .14 - x I 03 28 - MA P g 06 > / 2 1 .02 0 _r-,- a01 O9 l- O7 1_ oe 1 0~~~~~~ylcAPIo:K - Migration FIG. 2. Electrophoresis of 2 X-polymerized microtubule protein on a Laemmli (17) sodium dodecyl sulfate-polyacryl- amide gel, showing the resolution of MAP,, MAP2, tubulin 1, 2.0 and tubulin 2. 1.8 a' 1.6 NaCi, 0.01 M sodium phosphate, pH 7.4). Intracerebral injec- 2 1.4 were as 1 was tions performed described (8). After hr, the chick E 1.2 decapitated and 1 X-polymerized microtubule protein was 1.0 prepared as described above. The microtubule protein was O) subjected to sodium dodecyl sulfate-polyacrylamide gel IE0 electrophoresis (17); the gels were then scanned and sliced, and E 0.6 1H and 32p were determined by liquid scintillation counting. RESULTS When 2X -polymerized microtubule protein was incubated with [,y-32P]ATP, in the presence and absence of cyclic AMP, Time (min) and then subjected to sodium dodecyl sulfate-polyacrylamide .16 slab gel electrophoresis, a pattern of protein phosphorylation .14 of the type shown in Fig. 1 was observed. Several of the protein E .12 bands appearing on the gel were phosphorylated in the ab- 0) ca. sence of cyclic AMP, and the rate of their phosphorylation < .10 was stimulated by cyclic AMP. One protein of high molecular weight, designated microtubule-associated protein2 (MAP2), E .08 which constituted only 3% of the total protein on a weight ..06 basis, was found to contain 65% of the total incorporated 03 radioactive phosphate in the presence of cyclic AMP. The £ .04 relative molecular weights of microtubule-associated protein, E^.02, (MAP,) and MAP2 were determined to be approximately 0 350,000 and 300,000, respectively (data not shown), by com- 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 paring their migration with that of known protein standards pH on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (14). A comparison of the phosphorylation of MAP1, FIG. 3. Endogenous phosphorylation of MAP2 as a function of: cyclic AMP concentration (top); incubation time, in the MAP2, and tubulin is shown in Table 1. Although a measur- absence (0) or presence (0) of 10-5 M cyclic AMP (middle); able amount of 32p was incorporated into tubulin, the molar pH, in the absence (0) or presence (0) of 10-5 M cyclic AMP rate of incorporation of phosphate (mol of phosphate/mol of (bottom). Standard reaction conditions were used, except for the protein per min) into MAP2 was about 650 times greater than changes indicated. Buffers (50 mM) were: pH 4.5, sodium acetate; into tubulin. pH 5.3 and pH 6.2, sodium Mes; pH 6.8 and pH 7.4, sodium In order to determine whether MAP2 and the protein kinase Hepes; pH 8.0 and pH 8.5, Tris-HCl. co-purified with tubulins 1 and 2 during assembly-disassem- bly, the relative amounts of tubulin 1, tubulin 2, MAP1, and tion at each stage of the purification are listed in Table 2. MIAP2, as well as the initial rate of phosphorylation of MAP2, After the first two cycles of assembly-disassembly, the relative were measured for five cycles of assembly-disassembly. Fig. 2 amounts of the four major proteins remained constant, as did shows a representative photograph of a 2 X -polymerized the rate of phosphorylation of MAP2. These results indicate microtubule protein preparation separated on a polyacryl- that MAP2, and the protein kinase that catalyzed its phos- amide gel by the method of Laemmli (17), and the correspond- phorylation, co-purified with tubulins 1 and 2 during succes- ing absorbance scan, demonstrating the resolution of the four sive cycles of assembly-disassembly. major protein components observed in this system. The Some properties of the MAP2 phosphorylation reaction, relative amounts of these proteins and the rate of endogenous with a 2X-polymerized microtubule protein preparation, are phosphorylation of MAP2 in the microtubule protein prepara- shown in Fig. 3. A half-maximal increase in the rate of phos- Downloaded by guest on September 23, 2021 180 Biochemistry: Sloboda et al. Proc. Nat. Acad. Sci. USA 72 (1975) centration is shown in Fig. 4. The rate of phosphate incorpora- tion per mol of MAP2 increased with increasing protein con- centration over the range 0.1-2.0 mg/ml in the presence, but not in the absence, of cyclic AMP. The net result was an 0.8 _ enhancement of the effect of cyclic AMP with increasing E protein concentration. 0.6 To determine if MAP2 was phosphorylated in vivo, [32P]_ orthophosphate was injected intracerebrally into chick brains; O0 0.4 [8H]leucine was injected simultaneously as a marker for newly synthesized protein. The brains were removed 1 hr later; 1 X - co0.2 -o polymerized microtubule protein was prepared and subjected 1 to sodium dodecyl sulfate-polyacrylamide gel electrophoresis 0 0 0.5 1.0 1.5 2.0 2.5 3.0 (Fig. 5). All four major proteins on the gel, tubulin 1, tubulin Protein Concentration, mg/nr 2, MAP1, and MAP2, were labeled with tritium. However, FIG. 4. Endogenous phosphorylation of MAP1 as a function only MAP1 and MAP2 were significantly labeled with 82p. of microtubule protein concentration. Incubations were carried out under standard reaction conditions, in the absence (0) or DISCUSSION presence (0) of cyclic AMP, except for the variation in protein Preparations of microtubules assembled in vitro contain concentration. intrinsic protein kinase activity, as determined by their ability to phosphorylate an endogenous protein substrate (MAP2, phorylation of MAP2 was observed with 2 X 10-7 M cyclic Fig. 1). The rate of the endogenous phosphorylation reaction AMP (Fig. 3, top). Cyclic GMP (10lM) had no effect on the was markedly affected by cyclic AMP. Half-maximal stimula- phosphorylation of MAP2 (data not shown). In the presence of tion of MAP2 phosphorylation was obtained with 2 X 10-7 cyclic AMP, the incorporation of 32p was linear for about 3 M cyclic AMP. min and was maximal at 10 min (Fig. 3, middle). Cyclic Both the MAP2 and protein kinase appear to be integral AMP caused a 4-fold increase in the initial rate of phos- components of microtubule preparations assembled in vitro. phorylation of MAP2. The amount of 32p incorporated into The data showing that the ratio of MAP2 to tubulin remained MAP2 after a 45-min incubation was about 1.0 and 1.9 mol of constant through several cycles of assembly-disassembly phosphate per mol of MAP2, in the absence and presence of (Table 2) indicate that the association of MAP2 with micro- cyclic AMP, respectively. The pH dependence of the phos- tubules assembled in vitro is not a random phenomenon. phorylation reaction showed a broad maximum centered at Moreover, the data showing that the rate of phosphorylation about pH 6.8 (Fig. 3, bottom). In the absence of cyclic AMP, of MAP2 remained constant during purification through sev- the dependence of activity on pH was much less pronounced. eral cycles of assembly-disassembly (Table 2) indicate that The dependence of MAP2 phosphorylation on protein con- the association of the intrinsic protein kinase with micro-

.90-

.63 - cpm

A560 nm .45-

.27.

.18

.09. *50

1 (U Ou wU 40 .J,gn Inv (+) Slice Number (-) FIG. 5. Phosphorylation of \'IAP, and AIAP2 in vivo. One hour after intracerebral injection of [32Plorthophosphate and ['Hlleucine into chick brains, 1 X-polymerized microtubule protein was prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (17). The gel was stained with Coomassie blue, scanned at 560 nm, and sliced into 1-mm sections. The radioactivity was determined by scintillation counting. Solid line, Ao60; *, 3H; X, 32p. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Phosphorylation of Microtubule-Associated Protein 181

tubules assembled in vitro is also not a random phenomenon. This work was supported by U.S. Public Health Service Eipper (21) and Leterrier et al. (22) have presented evidence Grants MH-17387 and NS-08440 to P.G., U.S. Public Health Service Grant NS-10907 to J.L.R., and American Cancer Society that the protein having cyclic AMP-dependent protein kinase Grant VC-120 to J.L.R. S.A.R. is the recipient of a Postdoctoral activity is probably not the tubulins, in contrast to what had Fellowship 1-FO3-CA-54387 from the National Cancer Institute been previously proposed (3-5). of the National Institutes of Health. R.D.S. is supported by U.S. As shown in Table 1, MAP2 is 650 times more effective as a Public Health Service Grant GM-14642. substrate than the tubulins. One other protein, barely dis- 1. Goodman, D. B. P., Rasmussen, H., DiBella, F. & Guthrow, cernible in the protein-staining pattern in Fig. 1, incorporated C. E., Jr. (1970) Proc. Nat. Acad. Sci. USA 67, 652-659. about 20% of the total 32p appearing on the autoradiograph. 2. Lagnado, J. R., Lyons, C. A., Weller, M. & Phillipson, 0. This protein migrated with an apparent molecular weight of (1972) Biochem. J. 128, 95P. 73,000. The phosphorylation of this protein and that of tubulin 3. Soifer, D. (1972) J. Biol. 55, 245a. 4. Soifer, D. (1973) J. Gen. Physiol. 61, 265. were qualitatively similar to that of MAP2 with respect to 5. Soifer, D., Laszlo, A. H. & Scotto, J. M. (1972) Biochim. dependence on cyclic AMP concentration and pH. Biophys. Acta 271, 182-192. At present, the function of the observed phosphorylation of 6. Weisenberg, R. C. (1972) Science 177, 1104-1105. an endogenous substrate by an intrinsic cyclic AMP-depen- 7. Borisy, G. G. & Olmsted, J. B. (1972) Science 177, 1196- dent protein kinase in preparations of microtubules assembled 1197. in vitro is not known. The phosphorylation could affect the 8. Dentler, W. L., Granett, S., Witman, G. B. & Rosenbaum, Proc. Nat. initiation or elongation steps of microtubule formation or the J. L. (1974) Acad. Sci. USA 71, 1710-1714. disassembly of microtubules. Another possibility is that the 9. Erickson, H. P. (1974) J. Cell Biol. 60, 1,53-167. 10. Gaskin, F., Kramer, S. B., Cantor, C. R., Adelstein, R. & phosphorylation of MAP2 is related to the function of micro- Shelanski, M. L. (1974) FEBS Lett. 40, 281-286. tubules, rather than to the assembly process. 11. Burns, R. G. & Pollard, T. D. (1974) FEBS Lett. 40, 274- In this regard, it is interesting that the high molecular 280. weight proteins, MAP1 and MAP2, associated with neuro- 12. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. tubules assembled in vitro have some characteristics in com- Nat. Acad. Sci. USA 70, 765-768. mon with , the high molecular weight ATPase that 13. Post, R. L. & Sen, A. K. (1967) in Methods in Enzymology, eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, forms the arms of the flagellar outer doublet microtubules, and New York), Vol. X, pp. 773-776. that provides the motile force for flagellar movement (23-26). 14. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) MAP1 and MAP2 have mobilities on 3.5% sodium dodecyl Biochemistry 10, 2606-2617. sulfate-polyacrylamide gels similar (10, 11) but not identical 15. Johnson, E. M., Ueda, T., Maeno, H. & Greengard, P. (I. Tyler and J. L. Rosenbaum, unpublished observations) to (1972) J. Biol. Chem. 247, 5650-5652. those of the flagellar dynein proteins; moreover, it has recently 16. Ueda, T., Maeno, H. & Greengard, P. (1973) J. Biol. Chem. 248, 8295-8305. been reported (8, 10, 11) that MAP1 and MAP2 of neuro- 17. Laemmli, U. K. (1970) Nature 227, 680-685. tubules assembled in vitro have ATPase activity. Finally, both 18. Gorovsky, M. A., Carlson, K. & Rosenbaum, J. L. (1970) neurotubules assembled in vitro (8, 10) and neurotubules in Anal. Biochem. 35, 359-370. situ (27) have been shown to be covered with a filamentous 19. Schacterle, G. R. & Pollack, R. L. (1973) Anal. Biochem. 51, coating; if the tubules are assembled in the absence of MAP, 654-655. and MAP2 these filaments are absent from the surface of the 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. tubules (28), indicating that these filaments are composed of B. A. J. Biol. 1398-1406. MAP. is to that these filaments 21. Eipper, (1974) Chem. 249, It tempting speculate (MAPs) 22. Leterrier, J. F., Rappaport, L. & Nunez, J. (1974) Mol. associated with neurotubules may be motile proteins involved Cell. Endocrinol. 1, 65-75. in axoplasmic transport, and that their-phosphorylation in re- 23. Gibbons, I. R. (1965) Arch. Biol. 76, 317-352. sponse to cyclic AMP may be related to their motile function. 24. Summers, K. E. & Gibbons, I. R. (1971) Proc. Nat. Acad. The phosphorylation studies in vivo indicated that both Sci. USA 68, 3092-3096. MAP1 and MAP2 were phosphorylated, while very little 25. Gibbons, I. R. & Fronk, E. (1972) J. Cell Biol. 54, 365-381. 32p was associated with the tubulin (Fig. 5). Further phos- 26. Gibbons, B. H. & Gibbons, I. R. (1972) J. Cell Biol. 54, 75-97. phorylation experiments with brain slices, and with neuro- 27. Burton, P. R. & Fernandez, H. L. (1973) J. Cell Sci. 12, blastoma cells in tissue culture, should determine the possible 567-584. relationship of MAP phosphorylation to neurotubule assembly 28. Dentler, W. L., Granett, S. & Rosenbaum, J. L. (1975) J. or function. Cell Biol., in press. Downloaded by guest on September 23, 2021