Phosphorylation and activation of smooth muscle myosin by Acanthamoeba myosin I heavy chain kinase.

Acanthamoeba myosin I heavy chain kinase activates the actin-activated Mg2+ -ATPase activity of the Acanthamoeba myosin I isoenzymes, myosins IA and IB, by phosphorylating a single site within the myosin heavy chain. In this paper, we report that myosin I heavy chain kinase also phosphorylates isolated turkey gizzard smooth muscle myosin light chains, gizzard smooth muscle heavy meromyosin, and intact gizzard smooth muscle myosin, all in the absence of Ca2+ and with specific activities close to those measured for purified Ca2+/calmodulin-dependent gizzard smooth muscle myosin light chain kinase. Myosin I heavy chain kinase incorporates a maximum of 2 mol of phosphate/mol of heavy meromyosin, both by itself and together with smooth muscle myosin light chain kinase (the light chain kinase alone incorporates 1.6 mol of phosphate/mol of heavy meromyosin). Both kinases phosphorylate intact smooth muscle myosin to a maximum of 2 mol of phosphate/mol of myosin. Myosin I heavy chain kinase fully activates the actin-activated Mg2+ -ATPase of both myosin and heavy meromyosin. Two-dimensional tryptic peptide maps of isolated light chains phosphorylated by myosin I kinase show the same phosphopeptide as for light chains phosphorylated by the light chain kinase. These results support the conclusion that myosin I heavy chain kinase phosphorylates gizzard smooth muscle myosin at the same site within the 20,000-Da light chain as does smooth muscle myosin light chain kinase. The results suggest that the amino acid sequence around the phosphorylation site within the heavy chain of Acanthamoeba myosin I isoenzymes may be similar to the primary sequence around the phosphorylation site within the smooth muscle myosin light chain.

Acanthamoeba myosin I heavy chain kinase activates the actin-activated Mg2+-ATPase activity of the Acanthamoeba myosin I isoenzymes, myosins IA and IB, by phosphorylating a single site within the myosin heavy chain. In this paper, we report that myosin I heavy chain kinase also phosphorylates isolated turkey gizzard smooth muscle myosin light chains, gizzard smooth muscle heavy meromyosin, and intact gizzard smooth muscle myosin, all in the absence of Ca2+ and with specific activities close to those measured for purified Ca2+/calmodulin-dependent gizzard smooth muscle myosin light chain kinase. Myosin I heavy chain kinase incorporates a maximum of 2 mol of phosphate/ mol of heavy meromyosin, both by itself and together with smooth muscle myosin light chain kinase (the light chain kinase alone incorporates 1.6 mol of phosphate/ mol of heavy meromyosin). Both kinases phosphorylate intact smooth muscle myosin to a maximum of 2 mol of phosphate/mol of myosin. Myosin I heavy chain kinase fully activates the actin-activated M8+-ATPase of both myosin and heavy meromyosin. Two-dimensional tryptic peptide maps of isolated light chains phosphorylated by myosin I kinase show the same phosphopeptide as for light chains phosphorylated by the light chain kinase. These results support the conclusion that myosin I heavy chain kinase phosphorylates gizzard smooth muscle myosin at the same site within the 20,000-Da light chain as does smooth muscle myosin light chain kinase. The results suggest that the amino acid sequence around the phosphorylation site within the heavy chain of Acanthamoeba myosin I isoenzymes may be similar to the primary sequence around the phosphorylation site within the smooth muscle myosin light chain.
Myosin I heavy chain kinase has recently been purified to homogeneity from the soil amoeba, Acanthamoeba castellanii (1). Myosin I heavy chain kinase is an approximately globular protein containing one polypeptide of Mr = 107,000. The kinase phosphorylates the heavy chain, but not the light chain, of the single-headed Acanthamoeba myosin I isoenzymes, myosins IA and IB (1-3). Myosin I heavy chain kinase phosphorylates both myosin I isoenzymes at one site within the heavy chain, which has been shown to be a serine residue in the case of myosin IB (1). Maximal phosphorylation of myosin IA and IB results in a 20-fold increase in their actinactivated M$+-ATPase activities over the unphospho-* The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rylated enzymes (3,4). The purified kinase has a high specific activity toward myosin IA and IB, about 4 pmol of phosphate incorporated/min. mg (30 "C) at concentrations of myosin I that are well below saturating levels (1). The kinase requires Mg2+ but is not dependent on Ca2+, Ca2+/calmodulin, or CAMP for activity (1).
Investigation of myosin I heavy chain kinase substrate specificity revealed that the kinase also phosphorylates isolated turkey gizzard smooth muscle myosin light chains at a high rate (1, 5 ) . In this paper, we extend this observation, showing that myosin I heavy chain kinase phosphorylates intact gizzard smooth muscle myosin and heavy meromyosin at high rates, that myosin I heavy chain kinase fully activates the actin-activated Ma*+-ATPase activity of smooth muscle myosin and heavy meromyosin, and that this heavy chain kinase phosphorylates the same site within the 20,000-Da myosin light chain as does purified Ca*'/calmodulin-dependent gizzard smooth muscle myosin light chain kinase.

EXPERIMENTAL PROCEDURES
Proteins-Acanthamoeba myosin I heavy chain kinase was purified to greater than 95% homogeneity, as judged by SDS-PAGE,' by the procedure of Hammer et al. (1). Acanthurnoeba myosin IB was purified as described by Maruta et al. (6), as modified by Albanesi et al. (4). Rabbit skeletal muscle F-actin, purified according to Eisenherg and Kielley (7), was a gift of Dr. Lois Greene (National Institutes of Health).
Turkey gizzard smooth muscle myosin and heavy meromyosin were prepared according to 9). The 20,000-Da light chains of both proteins contained negligible phosphate, as shown by glycerol-urea gel electrophoresis. The heavy meromyosin used in the experiments described in Table I ,Fig. 1,and Fig. 2 was contaminated with a very small amount of both Ca*+/calmodulin-dependent and Ca2+-independent light chain kinase activities. After a 20-min incubation of heavy meromyosin alone (30 "C), approximately 0.1-0.15 mol of phosphate was incorporated/mol of heavy meromyosin in the presence of Ca2+/calmodulin and 0.05 mol of phosphate/mol of heavy meromyosin in the presence of EGTA. The heavy meromyosin used in the experiments described in Table 11, which was prepared by chymotryptic digestion in the presence of EGTA, was contaminated with a very small amount of Ca2+/calmodulin-dependent light chain kinase activity only (a 20-min incubation of heavy meromyosin alone at 30 "C yielded less than 0.1 mol of phosphate/mol of heavy meromyosin). Smooth muscle myosin was essentially devoid of devoid of phosphatase activity, since the phosphorylated enzymes did myosin kinase activity. Both myosin and heavy meromyosin were not lose detectable amounts of protein-hound phosphate after 1 week of storage at 4 "C. Phosphate-free turkey gizzard total smooth muscle light chains (6:4 ratio, 20,000-to 17,000-Da light chain) were prepared from purified myosin as described by Sellers et al. (10). Ca2+/calmodulin-dependent turkey gizzard smooth muscle myosin light chain kinase, purified by the method of Aldelstein and Klee ( l l ) , and porcine brain calmodulin, prepared according to Klee (12), were generous gifts of Dr. Elizabeth Payne (National Institutes of Health). Rabbit skeletal muscle heavy meromyosin, prepared by the method of Weeds and Pope (13) using a 2-min digestion with a 1:2000 (w/w) ratio of chymotrypsin to myosin, was a generous gift of Dr. Joseph Chalovich (National Institutes of Health). SDS-PAGE of skeletal muscle heavy meromyosin revealed greater than 90% intact light chains. Assay of Actin-activated Mg?"ATPase of Gizzard Smooth Muscle Myosin and Heavy Meromyosin-Myosin and heavy meromyosin were phosphorylated by myosin I heavy chain kinase or smooth muscle myosin light chain kinase in a reaction preceding the ATPase assay (see below). For heavy meromyosin, the ATPase assay was performed at 30 "C in a mixture containing 2 mM imidazole (pH 7.0), 1.8 mM MgC12, 0.1 mM EGTA, 1 mM [y3'P]ATP (5 pCi/wmol), and 0.1 mM dithiothreitol in a final volume of 0.6 ml. Where indicated, skeletal muscle F-actin was added to a final concentration of 1 mg/ ml. The assay was initiated by the addition of heavy meromyosin (120 pg in 25 pl) to the assay mixture pre-equilibrated at 30 "C. At 3 and 6 min, 250.~1 aliquots were removed and 32Pi released from [y-32P]ATP was measured as described by Pollard and Korn (14). In all cases, the rate of 32Pi release was the same at the two time points.
For myosin, the ATPase assay was performed at 25 "C in an assay mixture containing 50 mM KC1, 15 mM Tris (pH 7.0), 5 mM MgCI2, 0.1 mM EGTA, 1 mM [Y-~'P]ATP (50 FCilpmol), and 0.1 mM dithiothreitol in a final volume of 1 ml. Myosin and actin were pre-mixed in 0.5 M KC1 and diluted 1:lO by dropwise addition into the assay mixture (without ATP) to final concentrations of 0.15 and 0.5 mg/ ml, respectively. After gentle stirring for 30 s, the reaction was initiated by the addition of ATP. At 30-s intervals, 100-pl aliquots were removed and assayed for 32Pi release. The rate was obtained from the linear portion of the time course (usually from time 0 to 3 min). Both myosin I kinase and smooth muscle light chain were devoid of M%f-ATPase activity. Assays were initiated by the addition of substrate and then kinase to a reaction mixture pre-equilibrated to 30 "C. Aliquots of 40 p1 were removed at 30-s intervals and protein-bound 32P was determined by filter paper assay as described by Hammer et al. (1). The amount of kinase added was such that less than 15% of the total substrate was phosphorylated. Under these conditions, the incorporation of 32P into substrate was linear with time and proportional to the amount of kinase added. Control incubations containing only substrate showed little or no phosphorylation; any incorporation that occurred in the absence of kinase was subtracted from the rate measured in the presence of added kinase. Control reactions containing only kinase showed negligible phosphorylation. The maximal extent of phosphorylation of heavy meromyosin was also determined by filter paper assay but with higher concentrations of kinases.
Peptide Mapping-Two-dimensional cellulose thin layer maps of tryptic peptides of phosphorylated smooth muscle myosin light chains were produced by the method of Gracy (15), as modified by Cote et al. (16). Total isolated myosin light chains (70 pg), phosphorylated to near maximal extent by either myosin I heavy chain kinase, smooth muscle myosin light chain kinase, or both kinases together, were precipitated by addition of 50% trichloroacetic acid to a final concentration of 10%. The precipitate was collected by centrifugation in an Eppendorf Microfuge, washed three times with 2 ml of 10% trichloroacetic acid containing 50 g/liter of sodium pyrophosphate, washed two times with 2 ml of absolute ethanol, and dried under a stream of NP gas. The dried light chains were dissolved in 500 pl of 0.1 M NH4HC03 and digested with 7 pg of L-l-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington,No. 3740) for 14 h at 30 "C with gentle stirring. An additional 7 pg of trypsin was added and incubations continued for 4 h. The samples were lyophilized several times to remove the buffer, dissolved in 40 pl of pH 1.9 electrophoresis buffer (acetic acid/formic acid/water; 8290, v/v) and 5 pl spotted onto a thin layer cellulose-coated sheet (20 X 20 cm, The concentrations shown are for smooth muscle myosin, smooth muscle heavy meromyosin, and, in the case of isolated myosin light chains, the concentration is that of the 20,000-Da light chain which is present in a mixture of 20,000-and 17,000-Da light chains (6:4 ratio of 20,000-to 17,000-Da light chain).
MIHCK, myosin I heavy chain kinase; SMLCK, smooth muscle myosin light chain kinase; SMLC, isolated smooth muscle myosin light chains; ND, not determined HMM, smooth muscle heavy meromyosin; SMM, smooth muscle myosin. L -" a ""_ "-_ by guest on March 24, 2020 http://www.jbc.org/ Eastman Kodak) along with 1 pl of methyl green marker. Electrophoresis was performed on a Camag TLE unit at 1000 V (4 "C). The run was stopped when the methyl green had migrated to within 1 cm of the cathodic wick. Following air-drying, ascending chromatography in the second dimension was performed in 1-butanol/pyridine/acetic acid/water (5033:1040, v/v). Radioactive peptides were visualized by autoradiography.
Miscellaneous Methods-The protein concentrations of the following proteins were determined from their extinction coefficients, E a gizzard smooth muscle myosin, 5.4 cm-l; gizzard heavy meromyosin, 6.5 cm-l; gizzard myosin light chain kinase, 11.4 cm-l; actin 11.5 cm". The protein concentrations of Acantharnoeba myosin I isoenzymes and myosin I heavy chain kinase were determined using the colorimetric assay of Bradford (17), with bovine serum albumin as a standard. Discontinuous SDS-PAGE was performed as described by Laemmli (18), followed by Coomassie blue staining according to Fairbanks et al. (19). Gels were destained in 10% acetic acid. Glycerolurea gel electrophoresis was performed as described by Perrie and Perry (20). For autoradiography of 32P-labeled proteins and peptide maps, dried gels and thin layer sheets were exposed to x-ray film (Kodak X-Omat AR-2) beneath a n intensifying screen (DuPont, Cronex Lightning Plus). ATP, imidazole, and EGTA were purchased from Sigma and [ T -~~P J A T P was from New England Nuclear. All other chemicals were reagent grade.

Initial Rates of Phosphorylation of Isolated Smooth Muscle Myosin Light Chains, Smooth Muscle Heavy Meromyosin, ana! Intact Smooth Muscle Myosin by Myosin I Heavy Chain
Kinase-The sDecific activities of Durified mvosin I heavv chain kinase a n i purified Ca2+/calmodulin-dep&dent smootk  (Table I). Myosin I heavy chain kinase was approximately 70% as active as smooth muscle myosin light chain kinase toward isolated smooth muscle myosin light chains.
With heavy meromyosin as substrate, myosin I heavy chain kinase was approximately 95% as active as the myosin light chain kinase. In addition, myosin I heavy chain kinase phosphorylated intact smooth muscle myosin, which was present in the assay in a filamentous state, at an appreciable rate. As shown previously (l), myosin I heavy chain kinase is fully active in the absence of free Ca". In separate experiments (data not shown), smooth muscle myosin light chain kinase did not phosphorylate purified Acanthamoeba myosin IB (1.7 FM) and myosin I heavy chain kinase did not phosphorylate rabbit skeletal muscle heavy meromyosin (3 p~) . Fig. 1 shows that myosin I heavy chain kinase alone incorporated a maximum of 2 mol of phosphate/mol of heavy meromyosin. Smooth muscle myosin light chain kinase alone incorporated a maximum of 1.6 mol of phosphate/mol of heavy meromyosin. When the same amounts of myosin I kinase and myosin light chain kinase that caused maximal phosphorylation alone were incubated together with heavy meromyosin, 2 mol of phosphate/mol of heavy meromyosin was obtained. These results suggest that at least 80% of the phosphate incorporated by myosin I heavy chain kinase was incorporated into the same site within the 20,000-Da myosin light chain that is phosphorylated by Da; soybean trypsin inhibitor, 21,500 Da; lysozyme, 14,400 Da. The two high molecular mass bands seen in lanes 2-5 are the 130,000-Da heavy meromyosin heavy chain and a 65,000-Da doublet, which results from a second proteolytic cleavage of the myosin heavy chain. This second cleavage does not affect the activity of heavy meromyosin, which dissociates into the two 65,000-Da polypeptides only under denaturing conditions. Lanes 10-15, the maximally phosphorylated smooth muscle myosin (SMM) samples shown here are the same as samples E, F, and G in Table 11. Lanes 10-12, Coomassie blue-stained gel (5 pgllane). Lanes 13-15, autoradiogram of the same gel. The samples contained, in addition to the reaction mix described in Table 11, the following additions: 0.5 mM EGTA and 260 nM myosin I heavy chain kinase (lanes 10 and 13): 0.1 PM calmodulin (lanes 11 and 14); 0.1 p~ calmodulin and 14 nM smooth muscle myosin light chain kinase (lanes 12 and 15). by guest on March 24, 2020 http://www.jbc.org/ Downloaded from smooth muscle myosin light chain kinase. In addition, myosin I heavy chain kinase may have incorporated as much as 0.4 mol of phosphate/mol of heavy meromyosin into site(s) not phosphorylated by smooth muscle myosin light chain kinase. The fact that the myosin light chain kinase incorporated only 1.6 mol of phosphate/mol of heavy meromyosin, instead of the expected 2 mol of phosphate/mol of heavy meromyosin, was not due to the presence of phosphate on the 20,000-Da light chain of heavy meromyosin used as substrate, since glycerol-urea gel electrophoresis showed negligible phosphorylated 20,000-Da light chain in the purified heavy meromyosin. Rather, the lower stoichiometry with smooth muscle myosin light chain kinase was probably due to partial proteolytic cleavage of the 20,000-Da light chain during chymotryptic digestion of intact myosin to generate heavy meromyosin, rendering about 20% of the 20,000-Da light chains nonphosphorylatable by the myosin light chain kinase. Fig. 2 (lanes 1-9) shows an SDS-polyacrylamide gel and autoradiogram of heavy meromyosin phosphorylated to greater than 95% of maximum by myosin I heavy chain kinase alone and smooth muscle myosin light chain kinase alone. In both cases, essentially 100% of the radioactive phosphate was incorporated into the 20,000-Da myosin light chain. A small amount of radioactivity was incorporated by both kinases into an apparent proteolytic product of the 20,000-Da myosin light chain which migrated at about 18,500 Da. The Acanthamoeba myosin heavy chain kinase did not incorporate any phosphate into the heavy meromyosin heavy chain, indicating that the additional 0.4 mol of phosphate/mol of heavy meromyosin incorporated by myosin I heavy chain kinase over and above that incorporated by smooth muscle myosin light chain kinase was within the 20,000-Da myosin light chain. In experiments described below (see Table 11), incubation of smooth muscle myosin with high concentrations of either myosin I heavy chain kinase or smooth muscle myosin light chain kinase resulted in incorporation of 2 mol of phosphate/ mol of myosin, as determined by filter paper assay. In Fig. 2  (lanes 10-15), an SDS-polyacrylamide gel and autoradiogram of these myosin samples reveals that essentially 100% of the radioactive phosphate incorporated by myosin I heavy chain kinase, as well as by smooth muscle myosin light chain kinase, was into the 20,000-Da myosin light chain.

Stoichiometry of Phosphate Incorporation into Smooth Muscle Heavy Meromyosin and Smooth Muscle Myosin by Myosin I Heavy Chain Kinase-
Activation

Activation of the actin-activated M p -A T P a s e of smooth muscle myosin and heavy meromyosin by myosin I heavy chain kinase
Heavy meromyosin was phosphorylated by preincubation for 5 min at 30 "C under four conditions, each containing 14.4 p~ heavy meromyosin, 50 mM Tris (pH 7.3), 1 mM magnesium acetate, 0.2 mM CaCI2, 0.25 mM dithiothreitol, and 0.2 mM ATP (final volume, 60 pl) and either 0.5 mM EGTA (A), 0.5 mM EGTA and 180 nM myosin I heavy chain kinase (B), 0.1 p~ calmodulin (C), or 0.1 p~ calmodulin and 100 nM smooth muscle myosin light chain kinase (D). Following preincubation, the Me-ATPase activity of heavy meromyosin was assayed in duplicate in the presence and absence of 1 mg/ml of skeletal muscle F-actin as described under "Experimental Ptocedures." To determine the stoichiometry of phosphate incorporation into these samples, a portion of each preincubation was subjected to ted 20,000-Da light chain was estimated by densitometry as described The stoichiometry of phosphate incorporation into myosin was determined by filter paper assay as described in the text. Identical values were obtained before and after the dialysis step. Estimation of myosin M e -A T P a s e activity was performed as described in the text. chains were phosphorylated to near maximal extent by myosin I heavy chain kinase alone, smooth muscle myosin light chain kinase alone, and both kinases together. Phosphorylated myosin light chains were digested with trypsin, the peptides separated by two-dimensional cellulose thin layer mapping, and the radioactive peptides identified by autoradiography (Fig. 3). Myosin light chains phosphorylated by smooth muscle myosin light chain kinase alone (Fig. 3A) yielded one major radioactive peptide as expected. Two very minor radioactive peptides were also seen. Myosin light chains phosphorylated by myosin I heavy chain kinase alone (Fig. 3B) yielded one major radioactive peptide with a migration similar to the peptide in Fig. 3A. Two other radioactive peptides of lower intensity were also seen. When myosin light chains were phosphorylated simultaneously by both myosin I heavy chain kinase and smooth muscle myosin light chain kinase, again only one major radioactive peptide was observed (Fig. 3C). These results, therefore, support the conclusion that most of the phosphate incorporated by myosin I heavy chain kinase was in the same tryptic peptide as the phosphate incorporated by smooth muscle myosin light chain kinase. In addition, myosin I heavy chain kinase also incorporated a small but significant amount of radioactivity into two peptides not phosphorylated by smooth muscle myosin light chain kinase.

DISCUSSION
Previous work has shown that purified Acanthamoeba myosin I heavy chain kinase phosphorylates a single site within the heavy chain of Acanthamoeba myosin I isoenzymes, resulting in a 20-fold increase in their actin-activated Mg2" ATPase activities (1-3). Measurements of phosphorylation rates a t subsaturating myosin I concentrations indicate that the VmaX for myosin I heavy chain kinase toward myosin I would probably exceed 10 pmol/min.mg (1). More importantly, myosin I heavy chain kinase phosphorylates only the heavy chain of the myosin I isoenzymes of Acanthamoeba (1-3). Myosin I isoenzymes which have been maximally phosphorylated by myosin I heavy chain kinase do not contain detectable levels of light chain phosphate (1). However, the results reported here demonstrate that Acanthamoeba myosin I heavy chain kinase phosphorylates isolated turkey gizzard smooth muscle myosin 20,000-Da light chains, and the 20,000-Da light chains of smooth muscle heavy meromyosin and intact smooth muscle myosin, all in the absence of Ca2+ and at high rates. While insufficient kinetic data were obtained to determine K,,, and V,,, values, at the substrate concentra-tions tested, myosin I heavy chain kinase demonstrated specific activities which were very close to those measured for purified Ca2+/calmodulin-dependent smooth muscle myosin light chain kinase.
Myosin I heavy chain kinase appears to phosphorylate the same site within the 20,000-Da myosin light chain as does smooth muscle myosin light chain kinase. This conclusion is based on the fact that 1) phosphorylation of heavy meromyosin by myosin I heavy chain kinase and smooth muscle myosin light chain kinase is largely mutually exclusive, 2) that myosin I heavy chain kinase fully activates the actinactivated M<+-ATPase of both myosin and heavy meromyosin, and 3) that tryptic digestion of isolated smooth muscle myosin light chains phosphorylated by both kinases yields a single major phosphopeptide. While myosin light chains phosphorylated by smooth muscle myosin light chain kinase alone yielded only one tryptic phosphopeptide as expected, and myosin light chains phosphorylated by myosin I heavy chain kinase primarily yielded the same phosphopeptide, the peptide maps of the latter also showed two other significant radioactive peptides. These peptides may contain secondary sites phosphorylated by myosin I heavy chain kinase and could explain the additional 0.2 mol of phosphate incorporated/mol of light chain which myosin I heavy chain kinase incorporates into heavy meromyosin over and above smooth muscle myosin light chain kinase. The secondary radioactive peptides might have been derived from incomplete digestion, secondary cleavages, or peptides with variable oxidation states, but these explanations would require that the event occurred specifically in the myosin I kinase-phosphorylated peptides and not in the smooth muscle myosin light chain kinase-phosphorylated peptides. The amount of myosin I heavy chain kinase in the light chain phosphorylation reactions was too low to contribute significant amounts of radioactive peptides by way of myosin I kinase autophosphorylation (1).
In addition to myosin I heavy chain kinase, CAMP-dependent protein kinase has been reported (21) to phosphorylate isolated gizzard smooth muscle myosin 20,000-Da light chains in the same site as does smooth muscle myosin light chain kinase. However, unlike myosin I heavy chain kinase and smooth muscle myosin light chain kinase, CAMP-dependent protein kinase does not phosphorylate the 20,000-Da light chain in intact smooth muscle myosin (22).
The substrate specificity of protein kinases is thought to depend at least in part upon the primary sequence around the phosphorylation site, with specific amino acid side chains near the phosphorylated residue playing an essential role in recognition of the substrate by the kinase (for reviews, see Refs. 23 and 24). The fact that purified Acanthamoeba myosin I heavy chain kinase readily phosphorylates smooth muscle myosin as well as Acanthamoeba myosin I isoenzymes indicates a sequence homology around the phosphorylation site in the Acanthumoeba myosin I heavy chain and the smooth muscle myosin light chain. However, Ca*+/calmodulin-dependent smooth muscle myosin light chain kinase did not phosphorylate Acanthamoeba myosin IB, perhaps because of steric hindrance. At present, neither the myosin I heavy chain nor the turkey gizzard smooth muscle myosin fight chain phosphorylation site sequence is known, although the sequence of the 20,000-Da myosin light chain from turkey gizzard is probably very similar to the known sequence for the chicken gizzard myosin light chain, Lys-Ala-Thr-Ser(P)-Asn-Val-Phe-Ser (25). Smooth muscle myosin light chain kinase has been shown to phosphorylate readily a heptadecapeptide with a similar sequence around the phosphorylated serine: Ser-Ser-Lys-Thr-Thr-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser(P)-Asn-Val-Phe-Ser (26). Future work, including sequencing the Acanthamoeba myosin I heavy chain phosphorylation site and studies with synthetic phosphorylatable peptide substrates, should provide direct information concerning the specificity determinants for Acanthamoeba myosin I heavy chain kinase.