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Identification of the Calmodulin-Bindingdomain Of Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3187-3191, May 1985 Biochemistry Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase (protein phosphorylation/Ca2+-dependent enzyme regulation/synthetic peptide) DONALD K. BLUMENTHAL*t, Koji TAKIO*t, ARTHUR M. EDELMAN*t, HARRY CHARBONNEAUt, KOITI TITANIt, KENNETH A. WALSHt, AND EDWIN G. KREBS*tt *Howard Hughes Medical Institute, tDepartment of Pharmacology, and tDepartment of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195 Contributed by Edwin G. Krebs, January 28, 1985 ABSTRACT In the course of determining the primary modulin-dependent enzyme structure. One of the goals in structure of rabbit skeletal muscle myosin light chain kinase this endeavor was to identify the calmodulin-binding domain (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a of this enzyme. The sequence and preliminary characteriza- peptide fragment was obtained that appears to represent the tion of a peptide derived from MLCK that displays the prop- calmodulin-binding domain of this enzyme. Low concentra- erties expected of a calmodulin-binding domain are de- tions of the peptide inhibited calmodulin activation of MLCK scribed in this report. This information should prove useful (Ki 1 nM). The peptide was not associated with a catalytical- in understanding the evolutionary relationships of calmodu- ly active, calmodulin-independent form of MLCK that was ob- lin-dependent enzymes as well as the molecular mechanisms tained by limited proteolysis. The peptide is 27 residues in of calmodulin action. length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with METHODS any known protein sequence. The peptide contains one trypto- phanyl residue and a high percentage of basic and hydropho- MLCK was purified from fresh rabbit skeletal muscle by a bic residues, but no acidic or prolyl residues. Much of the se- procedure similar to that described by Nagamoto and Yagi quence has a high probability of forming a helix. A chemically (8), details of which will be publisl~ed elsewhere. The en- synthesized peptide has been prepared to study the interac- zyme was subjected to limited proteolysis in order to gener- tions of the peptide and calmodulin in more detail. The intrin- ate lower molecular weight forms that retained catalytic ac- sic tryptophan fluorescence of the synthetic peptide shows a tivity (unpublished data). With trypsin, a mixture of two significant enhancement (w45%) in the presence of Ca2' and forms with Mrs of approximately 60,000 and 40,000 by Na- calmodulin; fluorescence enhancement is maximal at a pep- DodSO4/PAGE was generated. The mixture, which con- tide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose tained nearly equal amounts of the two forms, was complete- affinity chromatography in the presence of 2 M urea indicates ly calmodulin-dependent and was termed T60/40. Evidence that the interaction of peptide and calmodulin is Ca2+-depen- was obtained that the Mr 60,000 form was an intermediate dent. The results of these studies indicate that the catalytic and form that could be further degraded to the Mr 40,000 form. A calmodulin-binding domains of MLCK represent distinct and calmodulin-independent form of MLCK was generated by separable regions of the protein. In addition, the results pro- limited proteolysis with chymotrypsin. This form had a Mr of vide a basis for future studies of the molecular and evolution- 35,000 by NaDodSO4/PAGE and showed identical catalytic ary details of calmodulin-dependent enzyme regulation. activity in the presence of either 2 mM EGTA or 0.2 mM Ca2' and 5 gM calmodulin. This calmodulin-independent Calmodulin is a Ca2+-binding protein that is ubiquitously dis- form of MLCK was denoted C35. Details of the preparation tributed and highly conserved throughout eukaryotic evolu- and properties of these proteolyzed forms of MLCK will be tion. Although it is known to regulate many Ca2+-dependent described elsewhere. CNBr digestions of intact MLCK, enzymes (recently reviewed in ref. 1), little is known about T60/40, and C35 were performed following reduction and S- the interactions of calmodulin with these proteins at the mo- carboxymethylation as described by Takio et al. (9). After lecular level. Myosin light chain kinase (MLCK; ATP:pro- Iyophilization, the CNBr digests were either dissolved in wa- tein phosphotransferase, EC 2.7.1.37) is one of the better ter to assay for MLCK inhibition or fractionated by a combi- characterized calmodulin-regulated enzymes (reviewed in nation of size exclusion and reversed-phase high-pressure refs. 2 and 3) and is found in both muscle and nonmuscle liquid chromatography (HPLC). Details of peptide isolation tissues. The enzyme occurs in tissue-specific forms, which and characterization will be presented elsewhere. vary widely in molecular weight, antigenic determinants, Calmodulin was prepared from bovine testis by using and enzymatic properties (3, 4). It catalyzes the phosphoryl- batchwise DEAE-cellulose (DE-52, Whatman) chromatogra- ation of a specific class of myosin light chain subunit, termed phy, phenyl-Sepharose (Sigma) chromatography (10), and the P-light chain (5). In smooth muscle this phosphorylation gel filtration chromatography (Bio-Gel A-0.5m, Bio-Rad). is required for initiation of contraction (reviewed in refs. 2 Mixed myosin light chains from rabbit skeletal muscle were and 6), whereas in skeletal muscle the phosphorylation ap- prepared as described (11). Standard solid-phase methods pears to modulate the degree of tension produced during iso- (12) were used for synthesizing the calmodulin-binding pep- metric contraction (3, 7). tide. The composition and sequence of the peptide were veri- A determination of the amino acid sequence of rabbit skel- fied by amino acid analysis, Edman degradations, and pro- etal muscle MLCK was undertaken by these several labora- ton NMR spectroscopy. [Y_32P]ATP was obtained from New tories as part of a long-range study of protein kinase and cal- England Nuclear. Calmodulin-binding activity of peptides and CNBr digests The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: MLCK, myosin light chain kinase; Mops, 4-morpho- in accordance with 18 U.S.C. §1734 solely to indicate this fact. linepropanesulfonic acid. 3187 Downloaded by guest on September 29, 2021 3188 Biochemistry: Blumenthal et al. Proc. NatL Acad Sci. USA 82 (1985) was determined by assaying for their ability to inhibit cal- modulin (13, 14). To establish whether loss of calmodulin modulin-dependent MLCK activity. MLCK activity was de- sensitivity was related to the loss of a calmodulin-binding termined as described (11). All reaction mixtures (50 ,ul final fragment, CNBr digests of various proteolyzed forms of volume) contained 200 AM CaCl2, 10 mM magnesium ace- MLCK were assayed for inhibitory activity (Fig. 1; see tate, 2 mM [y-32P]ATP (:200 cpm/pmol), 15 mM 2-mercap- Methods for description of nomenclature). The unfraction- toethanol, 50 mM 4-morpholinepropanesulfonic acid (Mops) ated CNBr digest of T60/40 (a form which retains calmodu- at pH 7.0. Concentrations of proteins and peptides are indi- lin dependence) was inhibitory at low concentrations (K, = cated in the figure legends. The following catalytic rates 1.3 nM). The CNBr digest of C35 (a form lacking calmodulin were determined for use in calculating apparent Ki values: dependence), however, showed a marked reduction (by a vo, the catalytic rate in the absence of any inhibitor or exoge- factor of 38) in the ability to inhibit (K, = 170 nM). These nously added calmodulin (sufficient endogenous calmodulin data are consistent with there being a high-affinity calmodu- was present in the myosin light chain preparation to give val- lin-binding fragment in CNBr digests of calmodulin-depen- ues for VO/Vmax of 0.3-0.4); v;, the catalytic rate in the pres- dent forms of MLCK (intact MLCK and T60/40), which is of ence of inhibitor and no exogenously added calmodulin; much lower affinity and/or quantity in CNBr digests of a Vmax9 the catalytic rate in the presence of 1 gM exogenously calmodulin-independent form of the enzyme (C35). added calmodulin. The apparent Ki value for a given inhibi- Assays of several isolated CNBr fragments (Fig. 2) indi- tor was calculated by using the equation: cated that one peptide, termed M13, exhibited nearly all of the inhibitory capacity (K, = 9 nM) of the unfractionated di- [I] KcaM gest. Two other fragments, M11-12 and M12, showed no in- hibitory ability. Peptide maps of CNBr digests of MLCK and (Vm,./Vi) - 1 1 Vmax/Vi - 1 C35 indicated that M13 was degraded to a much shorter form (Vm./VO) - 1 in C35, whereas fragment M12 was found in digests of both C35 and MLCK (unpublished observations). A peptide cor- where [I] is the inhibitor concentration and KCaM is the con- centration of calmodulin required for half-maximal activa- responding to the sequence of M13 (see Fig. 3 for sequence) but lacking a carboxyl terminus homoserine was synthesized nM was used for KCaM in all calculations). tion (a value of 1.0 and assayed for its ability to inhibit calmodulin-dependent The equation assumes simple competition for Ca4' *calmodu- lin between the inhibitor and MLCK. The Ki values stated in MLCK activity. This synthetic peptide showed the highest potency = 0.9 nM) of any material tested (data not the text were calculated only for 0.1 < vi/vo < 0.9. (Ki shown). The lower inhibitory potency of M13 as isolated from the CNBr digest was probably due to side reactions of RESULTS sensitive side chains (e.g., tryptophan oxidation, asparagine structure determination of rab- deamidation), which tend to occur under the relatively harsh In the course of the primary conditions of CNBr digestion and the subsequent purifica- was to know wheth- bit skeletal muscle MLCK, it of interest tion procedures.
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