Proc. Natl. Acad. Sci. USA Vol. 85, pp. 6337-6341, September 1988 Biochemistry Ca2'/calmodulin-dependent protein kinase II: Identification of threonine-286 as the autophosphorylation site in the a subunit associated with the generation of Ca2-independent activity GERALD THIEL*, ANDREW J. CZERNIK*, FRED GORELICKt, ANGUS C. NAIRN*, AND PAUL GREENGARD* *The Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021; and tDepartment of Medicine, Yale University School of Medicine, New Haven, CT 06510 Contributed by Paul Greengard, June 3, 1988 ABSTRACT Autophosphorylation of Ca2+/calmodulin- dependent protein kinase II converts the enzyme to a Ca2+- independent form. The time course for this conversion corre- lates with the autophosphorylation of a threonine residue located within a thermolytic phosphopeptide common to the a and (3/fl' subunits. In the present study, this site was identified in the a subunit. After autophosphorylation under conditions that produced near-maximal Ca2 -independent activity, the a and (3/ig' subunits were separated by NaDodSO4/PAGE, and the a subunit was cleaved with cyanogen bromide. The major phosphopeptide (CB-1), containing phosphothreonine as the only radiolabeled amino acid, was purified by reverse-phase high performance liquid chromatography and subjected to automated gas-phase Edman degradation. The sequence ob- tained, Xaa-Arg-Gln-Glu-Thr-Val-Asp-Xaa-Leu-Lys-Lys- Phe-Asn-Ala-Arg-Arg-Lys-Leu, represented the NH2-terminal 18 residues (residues 282-299) of a 26-amino acid cyanogen bromide peptide predicted from the deduced primary structure of the a subunit and contained a consensus sequence for Ca2 /calmodulin-dependent kinase II phosphorylation that included Thr-286. The sequences obtained for two phospho- peptides derived from secondary chymotryptic digestion of CB-1 confirmed that Thr-286 was the phosphorylated residue. Ca2 + /calmodulin (CaM)-dependent protein kinase II (Ca2 + / CaM kinase II) is a multifunctional protein kinase that phosphorylates several substrate proteins including synapsin I, microtubule-associated protein 2, glycogen synthase, and tyrosine hydroxylase (refs. 1-3; for review, see ref. 4). Isozymes of Ca2+ /CaM kinase II, which have been purified from rat brain (1-3) and a variety of other tissues (4), all consist of high molecular weight complexes comprised of subunits of Mr 50,000 (a) and Mr 60,000/58,000 (3/(3'). The subunits are autophosphorylated in vitro in the presence of Ca2+ /CaM and ATP, which results in the conversion of the enzyme to a Ca2 +-independent form (5-9). The generation of the Ca2+-independent form coincides with the autophos- phorylation of a threonine residue contained within a phos- phopeptide common to the a and 13/,B' subunits (10). The deduced amino acid sequences of the a (11, 12) and P/,B' (13, 14) subunits of rat brain Ca2' /CaM kinase II have been described. Based on the consensus phosphorylation site sequence recognized by Ca2+ /CaM kinase II in several substrates (15), a number of potential autophosphorylation sites are found in each subunit. Among these are Thr-286 (a subunit) and Thr-287 (J/1' subunit), which are adjacent to the putative CaM-binding domain present in each subunit. It has been speculated that these sites may be involved in the regulation of kinase activity (10, 11). In the present study, purified rat forebrain Ca2 +/CaM kinase II was autophosphorylated with [y-32P]ATP by using conditions that generate the Ca2"-independent form of the enzyme. The subunits were separated and the a subunit was digested with cyanogen bromide (CNBr). A single, major 32P-labeled phosphopeptide, purified by reverse-phase HPLC, was sequenced directly or was subjected to second- ary digestion with chymotrypsin, after which the chymotryp- tic phosphopeptides were repurified and sequenced. These results provide direct evidence that Thr-286 is the autophos- phorylation site in the a subunit of Ca2 + /CaM kinase II associated with the generation of Ca2'-independence. MATERIAL AND METHODS Materials. Ca2 +/CaM kinase II was purified from rat forebrain as described (3). Calmodulin was prepared (16) from frozen rabbit brains obtained from Pel-Freez. Synapsin I was prepared from bovine brain as described (17). [y- 32P]ATP was purchased from New England Nuclear. CNBr, triethylamine, and trifluoroacetic acid were purchased from Pierce. a-Chymotrypsin was purchased from Cooper Bio- medical (Malvern, PA). Thermolysin was purchased from Calbiochem-Behring. Reverse-phase HPLC columns were purchased from Rainin [Woburn, MA; Vydac C18 (45 x 25 cm) and Brownlee C4 (0.21 x 3 cm)] and from J. T. Baker [Phillipsburg, NJ; Bakerbond wide-pore C4 (0.45 x 25 cm)]. Plastic-backed cellulose thin-layer chromatographic plates were purchased from Kodak. P-81 phosphocellulose paper was purchased from Whatman (Hillsboro, OR). Autophosphorylation of Ca2+/CaM Kinase II. Autophos- phorylation of Ca2-/CaM kinase II was performed and measured essentially as described (10), except that final concentrations of 15 mM CaCl2, calmodulin at 100 ttg/ml, and 3 ;LM [y-32P]ATP (25 mCi/iumol; 1 Ci = 37 GBq) were used in the reaction mixture. For analytical experiments, Ca2+/CaM kinase II was autophosphorylated for 10 sec, 1 min, and 5 min and the effect of autophosphorylation on enzyme activity was determined by using bovine synapsin I as substrate (10). For the preparative scale experiment, 1 mg of purified enzyme was autophosphorylated under the conditions described above for 10 sec at 0C. CNBr Cleavage of Ca2"/CaM Kinase II. The 32P-labeled a and ,///' subunits of Ca2+/CaM kinase II were separated by NaDodSO4/PAGE in 10% polyacrylamide gels (18), local- ized by autoradiography of the unfixed, dried gel, and excised. Reswollen gel pieces were pooled, frozen with liquid N2, and then thawed and homogenized in 2 ml of 10 mM NH4HCO3/1 mM phenylmethylsulfonyl fluoride. The ho- mogenates were centrifuged at 15,000 x g for 15 min and the supernatants were lyophilized. The eluted protein was treated with chloroform/methanol (19) and traces of Na- Abbreviations: CaM, calmodulin; Ca2"/CaM kinase II, Ca2"/ calmodulin-dependent protein kinase II. 6337 The publication costs of this article were defrayed in part by page charge payment. 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