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Cyclic AMP-Dependent Endogenous Phosphorylation of a Microtubule-Associated Protein (Protein Phosphorylation/In Vitro Assembly) ROGER D

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Cyclic AMP-Dependent Endogenous Phosphorylation of a Microtubule-Associated Protein (Protein Phosphorylation/In Vitro Assembly) ROGER D Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 177-181, January 1975 Cyclic AMP-Dependent Endogenous Phosphorylation of a Microtubule-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 Microtubules 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 tubulin, 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 tubulins. 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.
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