Identification, Phosphorylation, and Dephosphorylation of a Second Site for Myosin Light Chain Kinase on the 20,000-Dalton Light Chain of Smooth Muscle Myosin*

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated J. Biol. Chern. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphoryla- tion and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with 0-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kCat/Km for serine 19 and thre- onine 18 for isolated light chains are 550 and 0.2 min" pc"', respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kCat/Km values are 22.5 and 7.5 min" g"' the digestion, 99% of the incorporated

Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin is accepted as an important component of the regulatory mechanism in smooth muscle (1,2). Under most experimental conditions the extent of incorporation is limited to 1 mol of phosphate/mol of light chain and the site of * This work was supported by National Institutes of Health Grants HL 23615 and HL 20984 (to D. J. H) and by National Institutes of Health Grant HL 21471 (to M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phosphorylation has been identified as serine 19 (3-5). Recently, however, phosphorylation at a second site has been found (6,7). Cole et al. (6) showed that double phosphorylation of the 20,000-dalton light chain could be observed only if the chicken gizzard myosin was prepared under certain conditions involving an overnight settling period. The identity of the kinase responsible for the phosphorylation of the second site was not established and it was found also that the additional phosphorylation did not increase actin-activated ATPase activity (6). The results from our laboratory (7) differed from those of Cole et al. (6) in several respects. Phosphorylation of the second light chain site was achieved at high concentrations of myosin light chain kinase (MLC kinase'), and no dependence on preparative procedures was observed. It was suggested that the enzyme responsible for the extra phosphorylation was in fact MLC kinase and in addition it was shown that phosphorylation of the second light chain site markedly increased the actin-activated ATPase activity of myosin (7). The second phosphorylation site was shown to be a threonine residue (7), in agreement with the results of Cole et al. (6), and threonines 9, 10, or 18 were suggested as possible locations.
In this article other results regarding the second phosphorylation site are presented. The second site of phosphorylation is identified as threonine 18 and it is shown that this threonine is distinct from that phosphorylated by protein kinase C (8). Kinetics of phosphorylation and dephosphorylation of threonine 18 for the isolated light chain and intact myosin also are presented and compared to those involving serine 19.

MATERIALS AND METHODS
The following procedures were used for the isolation of proteins: myosin (9), MLC kinase (IO) and myosin light chain phosphatase (11) from frozen turkey gizzards; calmodulin from frozen beef testes (10); the 20,000-dalton light chain from gizzard myosin (12); and protein kinase C (partially purified) from cow brain (13). A spontaneously active preparation of bovine aorta phosphatase (14) was kindly supplied by Dr. J. DiSalvo (University of Cincinnati). a-Chymotrypsin (Type 111) was obtained from Sigma, and trypsin (treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained from Worthington.
Phosphorylation assays for MLC kinase and protein kinase C were carried out as described previously (IO). Other conditions are given in the figure legends. Isolation of the peptide containing the second phosphorylation site was as follows: the isolated 20,000-dalton light chain (12 mg at 2 mg/ml) was phosphorylated in 50 mM KCl, 30 mM Tris-HC1 (pH 7.5), 1 mM MgC12, 0.6 mM [-p3'P]ATP (approximately 50 cpm/pmol), 0.1 mM CaC12, 30 pg/ml calmodulin, and 100 pg/ml MLC kinase at 25 "C for 30 min. The reaction was stopped by the addition of trichloroacetic acid to 0.5%. The extent of phosphate incorporation was 1.9 mol of phosphate/mol of light chain. The phosphorylated light chain was dialyzed against several changes of 50 mM KCl, 10 mM Tris-HC1 (pH 7.5) to remove [Y-~'P]ATP. Digestion of the phosphorylated light chain was carried out at 37 "C for 15 min with 0.1 mg/ml a-chymotrypsin in 50 mM Tris-HC1 (pH 7. using a Perkin-Elmer Series 4 HPLC system. The radioactive fractions were pooled (94% of applied radioactivity was recovered), lyophilized, dissolved in 1.5 ml of 0.1% trifluoroacetic acid (in HzO), applied to HPLC on a C18 reverse phase column (ODS 120T, 25 X 0.46 cm, Toyo Soda, Japan), and eluted with a linear gradient, 0.1% trifluoroacetic acid to 65% CH3CN, 0.1% trifluoroacetic acid. A peak containing 32P was eluted at approximately 23% CH3CN (see Fig.   1A). This contained approximately 95% of the applied radioactivity. The radioactive fractions were combined, lyophilized, dissolved in 1.5 ml of 0.1% trifluoroacetic acid, and reapplied to the above C18 column. Elution was achieved using two steps, initially of 10% CH3CN, 0.1% trifluoroacetic acid and subsequently of 15% CHsCN, 0.1% trifluoroacetic acid. The radioactivity was eluted in a single peak in the latter solvent (see Fig. 1B). The radioactive fractions were pooled, lyophilized, and submitted for amino acid analyses and sequence determinations (see Table I). A partial sequence was obtained for this a-chymotryptic peptide. The a-chymotryptic peptide was subjected to further hydrolysis with trypsin (to give the tryptic subpeptide, see Table I) as follows: 10 nmol of a-chymotryptic peptide in 0.5% NH4HC03 (pH 8.5) was digested for 16 h at 25 "C with 1% (by weight) trypsin. The hydrolysate was dried, dissolved in 50 pl of 0.1% trifluoroacetic acid, applied to HPLC on a C18 reverse phase column (LiChrosphere Si 100 RP 18, EM Reagents) and eluted with a linear CH3CN gradient. The radioactive peptide eluted at 14% CH3CN. The amino acid composition of this peptide was determined (see Table I).
Amino acid analyses were carried out using an instrument that employs single-column ion exchange separation, with detection by postcolumn derivatization with ninhydrin. Its sensitivity limit is about 1 nmol. Sequences were determined using both gas-phase and spinning-cup sequencers. The gas-phase sequencer (Applied Biosystems) was used with a load of about 1 nmol of peptide, and the phenylthiohydantoin derivatives were identified on a Hewlett-Packard Model 1090 HPLC, using a Zorbax Cyanopropylsilane column (250 X 4.6 nm). In an effort to determine the site(s) of phosphorylation, one-half of each sample from the Applied Biosystems sequencer was subjected to liquid scintillation counting. After a run was complete, the disc to which the sample was applied was also counted. Since the radioactive material (the peptide as well as the phosphothreonine and/or phosphoserine) remained bound to the disc in the gas-phase sequencer, the peptide (1 nmol) was also analyzed in a Beckman 890C sequencer using the 0.1 M Quadrol program. The fractions were dried, dissolved in 0.1 ml of methanol, and subjected to liquid scintillation counting.

TABLE I
Amino acid compositions and sequences of the phosphorylated peptides 10 2 0

Ac-S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-(a)
( a ) NHz sequence of the 20-kDa light chain: sequence established by Pearson et al. (5). ( b ) The a-chymotryptic peptide: the underlined residues and the locations of the phosphates (shown by *) were determined by sequencer analysis. The order of the Thr and Ser in the a-chymotryptic and tryptic peptides was assumed to coincide with the assignments of Pearson et al. (5). (c) The tryptic subpeptide: the sequence of this peptide is assumed, based upon the correspondence of its comDosition and residues 17-22 of the light chain.
The identification of phosphorylated amino acids was as described previously (7). Other analytical procedures are given by Walsh et al. (15).

RESULTS AND DISCUSSION
Identification of the Second Site of Phosphorylation-The isolated 20,000-dalton turkey gizzard light chain was phosphorylated by MLC kinase approximately to 2.0 mol of phosphate/mol of light chain and digested with a-chymotrypsin (see "Materials and Methods"). Under the conditions of proteolysis, the light chain is degraded to a 16,000-dalton fragment and the 32P is quantitatively released in a trichloroacetic acid-soluble peptide (7). The latter was applied initially to a gel filtration column (to remove [y3'P]ATP) and subsequently to reverse phase chromatography. As shown in Fig.  1A, the radioactivity is found predominantly in one peak. To achieve further purification, this fraction was reapplied to reverse phase chromatography and the elution profile is shown in Fig. 1B. A single peak was associated with the 32P label. The recovery of radioactivity at each step during this isolation procedure was greater than 90% and it is therefore reasonable to conclude that both phosphorylation sites are in the fraction shown in Fig. 1B. The amino acid composition of this fraction, shown in Table I, suggests that it is a pure peptide. The first 7 residues were identified as their phenylthiohydantoin derivatives, using the gas phase Applied Biosystems sequencer, and comparison of this sequence with the  sequence published by Pearson et al. placed this peptide unambiguously within the sequence of the light chain. The radioactive phosphate was expected in one or both of the next 2 residues, but no counts were observed when these (or any other steps) were counted, presumably because the phosphate groups bind to the sample disc in the gas-phase sequencer. Analysis with the Beckman 890C Sequencer yielded counts in both steps 8 and 9. The recovery was about 5%, and the relative amount in the two steps suggested that they were phosphorylated to approximately equal extent. This peptide was then digested with trypsin and the radioactive subpeptide was isolated by reverse phase chromatography (see "Materials and Methods"); its composition is shown in Table I. Based on the sequencer results, and the amino acid composition of two peptides, the complete sequence of the a-chymotryptic peptide must be as shown in Table I. It was previously determined that the second site of phosphorylation is a threonine residue (6,7) and the combined data prove that this residue is threonine 18.
Phosphorylation Kinetics of Serine 19 and Threonine 18-Previously it was shown that the phosphorylation of threonine is slower than that of serine 19 (7) and at levels of phosphorylation below 1 mol of phosphate/mol of light chain only phosphoserine is detected. Since the two sites are kinetically so distinct, it is possible to determine the rate constants for phosphorylation of each site. Time courses of phosphorylation of serine 19 are shown in Fig. 2A for isolated light chains and intact myosin. (Note that different concentrations of MLC kinase were used in each case. See figure legend.) The inset shows a semilogarithmic plot of these data. For the isolated light chains, a single exponential is adequate to fit the data but for intact myosin two exponentials are required (16, 17).  Fig. 2B. In contrast to the results shown in Fig. 2A, the two time courses can be fit by a single exponential indicating that phosphorylation of threonine 18 in both isolated light chains and intact myosin is a random process. Values of L t / K m for intact myosin and isolated light chains are 0.44 and 0.20 min" j"', respectively. Both values are considerably lower than those obtained for the phosphorylation of serine 19, but the difference is more marked when the double phosphorylation of the isolated light chain is considered.
Dephosphorylation of Serine 19 and Threonine 18"If the phosphorylation of threonine 18 is to have any physiological role in the regulation of smooth muscle activity, it must be subject to reversible phosphorylation and dephosphorylation. To test this possibility we examined the kinetics of dephosphorylation for doubly labeled light chains (-2 mol of P/mol of light chain) and intact myosin (-4 mol of P/mol of myosin).
The time courses of dephosphorylation are shown in Fig. 3A for intact myosin and in Fig. 3B for isolated light chains. From these data it appears that both threonine and serine residues are dephosphorylated. Complete dephosphorylation of the light chains and intact myosin was obtained at higher concentrations of phosphatase (data not shown). The amount of phosphatase used was the same on a molar basis for the intact myosin and the isolated light chains. Dephosphorylation of intact myosin is approximately 3.8 times faster than the dephosphorylation of isolated light chains. The insets show semilogarithmic plots of the time course data and in both cases a random dephosphorylation process is indicated (i.e. a single exponential). During the dephosphorylation of intact myosin, samples were taken at different times and analyzed (by autoradiograms of acid hydrolysates (7)) for phosphoserine and phosphothreonine.  is random and that the phosphatase has no apparent preference for the phosphorylated forms of threonine 18 or serine 19.
For the above experiments the phosphatase was prepared from turkey gizzard according to Onishi et al. (11); however, similar results were obtained using the spontaneously active bovine aorta phosphatase (14).
Phosphorylation of Intact Myosin by Protein Kinase C-It was shown previously (8) that the 20,000-dalton light chains of intact turkey gizzard myosin can be phosphorylated by protein kinase C to the extent of 1 mol of phosphate/mol of light chain and that the major site of phosphorylation is a threonine residue. An obvious concern was that threonine 18 may serve as a target for both MLC kinase and protein kinase C. To assess this possibility we measured the sequential phosphorylation of intact myosin by protein kinase C and by MLC kinase. As shown in Fig. 4, phosphorylation of myosin by protein, kinase C results in the incorporation of approximately 1.8 mol of P/mol of myosin. Addition of MLC kinase and calmodulin at the point indicated by the arrow results in further phosphorylation. At the lower MLC kinase concentration' (5 pg/ml) approximately 2 additional mol of phosphate are incorporated, consistent with the phosphorylation of serine 19. At a higher MLC kinase concentration (100 rg/ml) an additional 3.6 mol of phosphate are incorporated, i.e. phosphorylation of both serine 19 and threonine 18. It is concluded, therefore, that the threonine residue phosphorylated by protein kinase C is not threonine 18. The identity of the protein kinase C phosphorylation site is not established but it might be located close to the MLC kinase sites in the N-terminal region of the light chain. This suggestion is based on observations made on the sequential a-chymotryptic degradation of the light chain which yields initially an 18,000dalton fragment followed by a 16,000-dalton fragment. The transition from 18,000 to 16,000 involves the removal of Nterminal peptides (residues 1-22) and results in the loss of the two MLC kinase sites and the loss of the protein kinase C site. (A 17,000-dalton fragment is produced by tryptic hydrolysis and this retains all phosphorylation sites.) Possible phosphorylation sites for protein kinase C are therefore threonines 9 and 10. An interesting point of speculation is that phosphorylation of residues 18 and 19 by MLC kinase results in an increase of actin-activated ATPase activity (7) but the phosphorylation of adjacent residues, suggested as 9 or 10, causes a decrease in actin-activated ATPase activity (8).