The 204-kDa Smooth Muscle Myosin Heavy Chain Is Phosphorylated in Intact Cells by Casein Kinase II on a Serine near the Carboxyl Terminus*

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serinelthreonine kinases assayed, only cas- ein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myo- sin heavy chain to a significant extent in vitro. Two- dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile- Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val.The Ser* represents the Ser(POJ which is

Myosin is a contractile protein involved in a variety of motile events including muscle contraction (l), cell locomotion (2), and cell division (3,4). The conventional myosin molecule is composed of two heavy chain subunits df approximately 200 kDa each, which form a globular amino-terminal head region, and a rod-like coiled coil carboxyl-terminal tail. The globular head region is noncovalently associated with two pairs of light chains of 20 and 17 kDa.
Reversible phosphorylation of the heavy chain and/or light chain subunits of myosin occurs throughout nature. The nonmuscle and smooth muscle myosins of some invertebrates have been shown to be regulated by heavy chain phosphorylation. Phosphorylation of the heavy chains of Dictyostelium and Acanthamoeba myosin inhibits the actin-activated Mg-* 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 USC. Section 1734 solely to indicate this fact.
ATPase activity and affects its ability to form stable bipolar filaments (5)(6)(7)(8). The myosin heavy chains from a molluscan smooth muscle can also be phosphorylated, and this modification may control the transition from a long lived state called "catch" to the relaxed state (9).
Vertebrate nonmuscle and smooth muscle myosin are regulated by phosphorylation of the 20-kDa light chains (LC&).' Myosin light chain kinase phosphorylation of LC,, in vitro allows the myosin Mg-ATPase to be activated by actin and favors myosin polymerization into filaments (1, 10,ll). Studies performed in situ suggest that LCZO phosphorylation is required for the initiation of contraction (1,10,11). The heavy chains of vertebrate nonmuscle (12-20) and smooth muscle myosin (21) can also be phosphorylated although the biological effects of these phosphorylations are not yet known. The identification of myosin heavy chain kinases in Acanthamoeba and Dictyostelium and myosin light chain kinases in vertebrate nonmuscle and smooth muscle cells and the localization of their phosphorylation sites have been essential to understanding the effects of phosphorylation on the regulation of myosin activity (1,22). Likewise, this information would help to elucidate the role of heavy chain phosphorylation in vertebrate nonmuscle and smooth muscle myosin function. Several heavy chain kinases and in some cases the sites of phosphorylation have been identified for vertebrate nonmuscle myosins. A calcium/calmodulin-dependent kinase has been shown to phosphorylate both intestinal brush-border epithelial cell myosin heavy chains (18) as well as the heavy chains of brain myosin (17). Casein kinase II has also been shown to phosphorylate brain myosin heavy chains (14), and recently the site of phosphorylation in brain myosin was identified within an amino acid sequence in the tail region of the molecule (23). The heavy chains of human platelet and rat basophil myosin have been shown to be phosphorylated by protein kinase C in vitro (19) and in intact cells (19,20), and the site of phosphorylation was localized to a serine residue in the carboxyl-terminal region of the heavy chain (24). To date, the myosin heavy chain kinase(s) catalyzing phosphorylation of the myosin heavy chain in vertebrate smooth muscle cells has not been identified, nor have the sites of heavy chain phosphorylation been determined. Two isoforms of myosin heavy chains have been identified in smooth muscle cells which differ in their amino acid sequence in the carboxyl-terminal tail regions (25). These isoforms (204 and 200 kDa) are generated from a single gene through alternative mRNA splicing. The functional significance of these two isoforms is not yet known although their expression appears to be developmentally regulated in the vascular system (26). In this report, we provide the first direct evidence that casein kinase II, a kinase believed to play a role in regulating cellular growth (27)

Phosphorylation of Smooth Muscle Myosin Heavy Chains in
Cultured Bovine Aortic Cells-Using polyclonal antibodies prepared against bovine aortic smooth muscle myosin, we immunoprecipitated myosin from first passage cultures of bovine aortic smooth muscle cells labeled metabolically with ['"Plorthophosphate. Following SDS-PAGE (12.5% gel) and autoradiography, we observed that '*P had been incorporated into both the myosin heavy chain and the 20-kDa light chain (Fig. lB, lane IA), in agreement with a previous report using ratlaorta (21). Closer analysis of the myosin heavy chain immunoprecipitate on SDS-polyacrylamide gels (5%) revealed one major and one minor myosin heavy chain band by Coomassie Blue staining (Fig. lA, lane 2). The major band was identified as smooth muscle myosin by immunoblotting using antibodies specific for smooth muscle and nonmuscle myosin (results not shown). We further identified the major band as the higher molecular weight isoform of the smooth muscle myosin heavy chain (MHC,), based on its mobility in SDS-polyacrylamide gels (5%) compared with purified aortic smooth muscle myosin (see below, Fig. 4, lane 2). The autoradiogram of the SDS-polyacrylamide gel (5%) of myosin heavy chains from cultured aortic cells (Fig. lB, lane 2A) showed incorporation of 32P into the smooth muscle MHC1. The minor band just below the smooth muscle MHCl band, seen on Coomassie Blue-stained gels and autoradiography (Fig. lA,   not present in immunoprecipitates if ATP was included in the immunoprecipitation buffer, indicating that it is co-precipitated and not immunoprecipitated.

Phosphorylation of Smooth Muscle Myosin Heavy Chains in
Vitro-Having established that the smooth muscle myosin heavy chain was phosphorylated in cultured bovine aortic smooth muscle cells, we next examined the ability of a variety of purified kinases to catalyze the phosphorylation of bovine aortic smooth muscle myosin in vitro. Myosin was incubated with kinase either after treatment with bacterial alkaline phosphatase or without prior phosphatase treatment (Table  I, dephosphorylated and untreated myosin, respectively). Of the several enzymes tested, only protein kinase C, CaM kinase II, and casein kinase II were able to phosphorylate the heavy chains of untreated myosin (Table I). CAMP-and cGMPdependent protein kinase did not catalyze the phosphorylation of myosin heavy chains or light chains. When myosin was treated with bacterial alkaline phosphatase prior to kinase addition, a 3-fold increase in phosphorylation of the heavy chain by casein kinase II to 0.6 mol/mol and a 2-fold increase in phosphorylation by CaM kinase II to 0.6 mol/mol was observed. The stoichiometry of phosphorylation with protein kinase C did not increase with dephosphorylated myosin as the substrate. These results suggest that myosin is already phosphorylated by casein kinase II or CaM kinase II in intact tissue or that it becomes phosphorylated during the purification process.
Phosphorylation of myosin by casein kinase II was specific for the heavy chain whereas CaM kinase II and protein kinase C also phosphorylated the LCZO subunit (Table I). The protein kinase C phosphorylation sites on the 20-kDa light chains have been identified as serine 1 and 2 and threonine 9 (38,39). Two-dimensional tryptic peptide mapping revealed that CaM kinase II phosphorylates LCZO on serine 19, one of the sites phosphorylated by myosin light chain kinase.*

Identification of the Smooth Muscle Myosin Heavy Chain
Kinase in Cultured Cells-We next determined whether the region of the smooth muscle myosin heavy chain that was phosphorylated in cultured cells corresponded to that phosphorylated in vitro by either protein kinase C, CaM kinase II, or casein kinase II. Two-dimensional tryptic peptide maps of myosin heavy chains phosphorylated either in cultured cells or in vitro by protein kinase C, CaM kinase II, or casein kinase II were compared. One major and one minor phosphopeptide were generated following tryptic digestion of 32Plabeled smooth muscle myosin heavy chains immunoprecipitated from cultured aortic smooth muscle cells (Fig. 2 identified on two-dimensional maps of tryptic digests from smooth muscle myosin heavy chains phosphorylated with casein kinase II in vitro (Fig. 2, panel 2). We confirmed that the peptides were the same by co-mapping the tryptic digests of myosin heavy chains phosphorylated with casein kinase II in vitro and those phosphorylated in cultured cells (Fig. 2,  panel 3). Of note, two-dimensional tryptic phosphopeptide maps of "*P-labeled smooth muscle myosin heavy chains immunoprecipitated from cultured bovine retinal pericytes were identical to those in Fig. 2 (results not shown). These results suggest that casein kinase II phosphorylation of smooth muscle myosin heavy chains is not limited to bovine aortic smooth muscle cells. Two-dimensional tryptic phosphopeptide maps of myosin heavy chains phosphorylated with protein kinase C revealed numerous (>lO) phosphopeptides (results not shown). Because the stoichiometry of phosphorylation of both untreated and dephosphorylated myosin was low, and the phosphate was distributed among several sites not phosphorylated in intact cells, we conclude that protein kinase C is not responsible for the myosin heavy chain phosphorylation observed in cultured cells. The peptide maps of myosin heavy chains phosphorylated with CaM kinase II contained one major and three minor phosphopeptides. The pattern, however, was completely different from that obtained with heavy chains from cultured cells (results not shown).

Phosphoamino
Acid Analysis of Myosin Heavy Chains-The phosphoamino acid content of the common peptides from myosin heavy chains phosphorylated in cultured cells or in vitro with casein kinase II was determined. Fig. 3 shows the results from the in vitro phosphorylation with casein kinase II and establishes that both peptides contain phosphoserine with no detectable amounts of either phosphothreonine or phosphotyrosine. Identical results (not shown) were obtained with phosphopeptides from myosin heavy chains labeled in cultured cells. The column was run as described under "Experimental Procedures." Arrows represent the changes in pH for the column washes and elution buffer. Radioactivity was detected by Cerenkov counting of the fractions (circles). Peptides were detected by A2s0 (squares). myosin by immunoblots using anti-smooth muscle myosin antibodies and by lack of cross-reactivity with antiplatelet myosin antibodies. By densitometry, we estimated that the relative abundance of the isoforms was equal although in cultured cells only MHC, is present (see Fig. 1). The distribution of "P in the two isoforms is shown in Fig. 4B, lane 2A.

Densitometry
of these bands showed that of the total s*P incorporated into heavy chains, greater than 85% was incorporated into MHC1.

Identification of the Casein Kinase II Phosphorylation
Site-Tryptic peptides of smooth muscle myosin heavy chains phosphorylated in vitro with casein kinase II were separated by Fe"'-iminodiacetic-Sepharose affinity column chromatography (Fig. 5). The tryptic digest was applied to the column in 0.1 M acetic acid, pH 2.0, unphosphorylated peptides were eluted by stepwise washing at acidic pH, and phosphorylated peptides were eluted at pH 8.3. Fractions 44-54 (77% of the total radioactivity applied) were pooled and applied to reverse phase HPLC. Four radioactive peaks were eluted (Fig. 6 The column was equilibrated with 10% acetonitrile, 0.1% trifluoroacetic acid in water and developed with an acetonitrile gradient of lo-60% in 0.1% trifluoroacetic acid (broken line). The flow rate was 1.0 ml/min, and l.O-min fractions were collected. Radioactivity was determined by Cerenkov counting of the fractions (solid line). Val 12 " 350 pmol was applied to the amino acid sequenator. B, C, and D) with an overall "'P recovery of 86%. Pooled fractions from the peaks were analyzed for amino acid composition and amino acid sequence. Peaks A and D had no absorption at 220 nm, contained no interpretable amino acid sequence, and therefore were not examined further. Peaks B and C, which were combined, gave the results presented in Table II. The sequence through the first 12 steps was Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser-Glu-Glu-Glu-Val. The amino acid composition of this fraction was consistent with this sequence plus the amino acids Asp-Ala-Arg.
To determine the exact location of this phosphopeptide in the smooth muscle MHC1, we compared the sequence with the published carboxyl-terminal sequence of the MHCl and MHC2 isoforms of rabbit uterine smooth muscle myosin (25). We recognized our peptide sequence within a predicted tryptic peptide sequence of rabbit MHC1, as shown between the vertical arrows in Fig. 7. This sequence is not present in MHC,. We demonstrated above (Fig. 3) that casein kinase II phosphorylates a serine residue in the smooth muscle MHC,.
Since the tryptic phosphopeptide we isolated as well as the corresponding tryptic peptide shown in Fig. 7 contain a single serine residue, we conclude that this is the location of the serine residue phosphorylated by casein kinase II in the MHC, isoform of bovine aortic smooth muscle myosin. DISCUSSION In the present study we found that casein kinase II, CaM kinase II, and protein kinase C catalyzed the phosphorylation of smooth muscle myosin heavy chains in uitro although the stoichiometry of phosphorylation with protein kinase C was The amino acid sequence of the casein kinase II phosphorylated tryptic peptide, VZENADGSEEEVDAR, in MHCl of bovine aortic smooth muscle is identical with an expected tryptic peptide, delineated by the arrows, in the carboxyl-terminal tail of rabbit uterine smooth muscle MHC, (25). This region is absent in rabbit uterine MHC,. Casein kinase II phosphorylated the serine marked with an asterisk in the bovine aortic sequence. The amino acid sequence and casein kinase II phosphorylation site of this tryptic peptide are also highly conserved in rat aortic MHC, and are absent from rat aortic MHCz (46 (44,45). The serine in our peptide is just so located. We confirmed the carboxyl-terminal location of the phosphorylated serine after identifying a sequence in the rabbit uterine smooth muscle MHCl which was identical to our phosphopeptide. Specifically, the site of phosphorylation was found to be within the nonhelical tail of MHC. We looked for conservation of the casein kinase II phosphorylation site in smooth muscle and nonmuscle myosins. Fig. 7 compares the carboxyl-terminal amino acid sequence of the smooth muscle myosin heavy chains for rabbit uterine MHC, and MHC, (25), rat aortic MHC and MHC2 (46), and the nonmuscle myosin heavy chains of human macrophage myosin (47), chicken intestinal epithelial cell myosin (48), and the amino acid sequence surrounding the site phosphorylated by casein kinase II in bovine brain myosin (23). The 6 amino acids shown in enlarged print within the box are exceptional in being highly conserved among smooth muscle and nonmuscle myosins. The work reported herein as well as previous work reported by others (23) indicate that the serine residues, shown within the box, in both the bovine aortic smooth muscle MHCl and the brain myosin heavy chain are substrates for casein kinase II. The boxed acidic amino acids carboxyl-terminal to the phosphorylated serines constitute a conserved consensus sequence for casein kinase II phosphorylation.
We suspect that the analogous serines in the rat aortic smooth muscle MHCi, the human macrophage myosin, and the chicken intestinal epithelial cell myosin will also serve as substrates for this enzyme. It is likely that additional smooth muscle MHC!, and nonmuscle myosin heavy chain isoforms from a variety of species and tissues will contain this conserved casein kinase II phosphorylation site. However, it is noteworthy that in the embryonic chicken gizzard amino acid sequence (not shown), the phosphorylatable serine is replaced by a glycine (49).
The functional consequences of heavy chain phosphorylation by casein kinase II are not yet known. The conservation by guest on March 23, 2020 http://www.jbc.org/ Downloaded from of amino acid sequence in an otherwise divergent region suggests that casein kinase II phosphorylation is important in myosin function and that smooth muscle MHCl and nonmuscle myosin may be regulated similarly. Moreover, the phosphorylation of MHC, and not MHC2 implies that these isoforms are regulated differently.
This area of sequence similarity is within a region of myosin which is required for assembly into filaments (50)(51)(52)(53), and phosphorylation of some myosin isoforms in this region has been shown to interfere with filament formation (6,8). There is to date no evidence that casein kinase II phosphorylation has an effect on filament formation.
MHC, phosphorylation by casein kinase II may be important in smooth muscle cell proliferation.
Casein kinase II phosphorylates a number of substrates that are critical in regulating cellular proliferation (44,45), and its activity may be modulated by growth factors (54)(55)(56)(57). Although the turnover rate of smooth muscle cells in the normal adult vasculature is low, in certain pathological conditions, such as atherosclerosis and hypertension, smooth muscle cells proliferate rapidly. Smooth muscle cells also proliferate rapidly during embryonic development and when placed into tissue culture. MHC, is selectively expressed over MHC:! during early embryonic development of the aorta (26) and in cultured cells (see Fig. 1) (41,42). Previous work has demonstrated that myosin heavy chain phosphorylation is high in cultured smooth muscle cells (21) but low in mature smooth muscle tissue (40). Thus, casein kinase II phosphorylation of MHCi may play a role in proliferating smooth muscle cells both in uiuo and in culture. The results reported here should facilitate studies on the function of smooth muscle myosin heavy chain phosphorylation.