Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells

Previous studies have demonstrated that rat aortic smooth muscle cells (SMC) show marked changes in smooth muscle (SM) alpha-actin content and fractional synthesis as a function of cell density and growth (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352; Blank, R., Thompson, M. M., and Owens, G. K. (1988) J. Cell Biol. 107, 299-306). Results of this study show that, although there is a 6-fold increase in SM alpha-actin content in postconfluent density arrested cultures as compared to proliferating subconfluent cultures, SM alpha-actin mRNA levels are not different between these cells. This suggests that the SM alpha-actin gene is constitutively active under both of these conditions and that accumulation of SM alpha-actin in postconfluent cells is due to translational and/or post-translational controls. The relationship between growth and cytodifferentiation was further explored by examining the effects of platelet-derived growth factor (PDGF)- or serum-induced growth on actin expression in postconfluent, quiescent cultures maintained in a defined serum-free media. Although both factors have been shown to stimulate proliferation and decrease fractional SM alpha-actin synthesis (Blank et al., 1988), their effects on actin mRNA levels were quite different. PDGF was found to induce a dramatic drop in SM alpha-actin steady state mRNA level but had no effect on nonmuscle beta-actin mRNA level. In contrast, serum stimulation was shown to increase nonmuscle beta-actin mRNA level, whereas SM alpha-actin mRNA level remained constant. Taken together these results indicate that PDGF is a specific and potent repressor of SM alpha-actin expression in vascular SMC and implicate a possible developmental role for PDGF in control of SMC differentiation. In addition, the observation that the level of SM alpha-actin mRNA is unaltered in serum-stimulated cells indicates that an absolute decrease in SM alpha-actin mRNA is not obligatory for cell cycle entrance.     

Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.     

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.     

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.     

cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.     

The heavy chain of smooth muscle myosin is phosphorylated in aorta cells

The 204-kDa smooth muscle myosin heavy chain (MHC) from rat aorta smooth muscle cells was found to be phosphorylated following isolation of myosin from strips of intact aorta as well as from primary cultures of aorta cells. Two-dimensional maps of the tryptic peptides revealed that the phosphate was confined to only three peptides and gave a similar pattern for the MHC isolated from intact aorta strips and cultured cells. This map was quite different from the phosphopeptide map found for the 196-kDa MHC of nonmuscle myosin isolated from the same cell culture. Smooth muscle MHC purified from primary cell cultures was found to contain approximately 0.7 mol of phosphate/mol of MHC while the nonmuscle MHC contained approximately 0.8 mol of phosphate/mol of MHC. These observations raise the possibility of an additional regulatory mechanism in smooth muscle operating via MHC phosphorylation.     

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.     

Maturational differences between vascular and bladder smooth muscle during ovine development

Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compared by quantifying contractile protein, myosin heavy chain (MHC) isoform contents, and contractile properties of aortas and bladders from female fetal (n = 19) and postnatal (n = 21) sheep. Actin, myosin, and protein contents rose progressively throughout development in both tissues (P 相似文献   

Myosin II isoforms in smooth muscle: heterogeneity and function

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC(20) has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ.     

Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation

The relationship between growth and cytodifferentiation was studied in cultured rat aortic smooth muscle cells (SMCs) using expression of the smooth muscle (SM)-specific isoactins (Vanderkerckhove, J., and K. Weber, 1979, Differentiation, 14:123-133) as a marker for differentiation in these cells. Isoactin expression was evaluated by: (a) measurements of fractional isoactin content and synthesis ([35S]methionine incorporation) by densitometric evaluation of two-dimensional isoelectric focusing sodium dodecyl sulfate gels, and (b) immunocytological examination using SM-specific isoactin antibodies. Results showed the following: (a) Loss of alpha-SM isoactin was not a prerequisite for initiation of cellular proliferation in primary cultures of rat aortic SMCs. (b) alpha-SM isoactin synthesis and content were low in subconfluent log phase growth cells but increased nearly threefold in density-arrested postconfluent cells. Conversely, beta-nonmuscle actin synthesis and content were higher in rapidly dividing subconfluent cultures than in quiescent postconfluent cultures. These changes were observed in primary and subpassaged cultures. (c) alpha-SM actin synthesis was increased by growth arrest of sparse cultures in serum-free medium (SFM; Libby, P., and K. V. O'Brien, 1983, J. Cell. Physiol., 115:217-223) but reached levels equivalent to density-arrested cells only after extended periods in SFM (i.e., greater than 5 d). (d) SFM did not further augment alpha-SM actin synthesis in postconfluent SMC cultures. (e) Serum stimulation of cells that had been growth-arrested in SFM resulted in a dramatic decrease in alpha-SM actin synthesis that preceded the onset of cellular proliferation. These findings demonstrate that cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state and indicate that growth arrest promotes cytodifferentiation in these cells.     

Segregated assembly of muscle myosin expressed in nonmuscle cells.

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.     

Smooth muscle myosin is composed of homodimeric heavy chains.

Vertebrate smooth muscle myosin heavy chains (MHCs) exist as two isoforms with molecular masses of 204 and 200 kDa (MHC204 and MHC200) that are generated from a single gene by alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). A dimer of two MHCs associated with two pairs of myosin light chains forms a functional myosin molecule. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHC204 were generated and used to immunoprecipitate purified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. MHC204 quantitatively removed from this mixture was completely free of MHC200. Immunoprecipitation of the supernatant with an antiserum that recognizes both isoforms equally well revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. A method is described for the purification of enzymatically active MHC204 and myosin on a protein G-agarose high performance liquid chromatography column containing immobilized MHC204 antibodies. We show, using an in vitro motility assay, that the movement of actin filaments by myosin containing 204-kDa heavy chains (0.435 +/- 0.115 microns/s) was not significantly different from that of myosin containing 200-kDa heavy chains (0.361 +/- 0.078 microns/s) or from myosin containing equal amounts of each heavy chain isoform (0.347 +/- 0.082 microns/s).     

Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle

To describe phenotypic changes of human aortic smooth muscle cells (SMCs), proportion of smooth muscle and nonmuscle variants of actin, myosin heavy chains (MHCs), vinculin, and caldesmon, during prenatal and several months of postnatal development was determined. In aortic SMCs from 9-10-week-old fetus, both nonmuscle and smooth muscle-specific variants of all four proteins were present, however, the nonmuscle forms were more abundant. During development, a shift towards the expression of muscle-specific variants was observed, although the time course of changes in protein variant content was not similar for all the proteins studied. By the 24th week of gestation, fractional content of alpha-smooth muscle actin and smooth muscle MHCs was rather close to that in the mature SMCs, and comprised approximately 80 and 90%, respectively, of the levels characteristic of SMCs from adult aortic media. On the contrary, fractional ratio of meta-vinculin and 150-kDa caldesmon was still rather low in the aorta from the 24-week-old fetus, did not increase in a 2-month-old child aorta, and did not reach the level characteristic of mature SMCs even in the 6-month-old child aorta. Thus changes in alpha-smooth muscle actin and smooth muscle MHC fractional content occur mainly during the prenatal period of development, before the 24th week of gestation; while meta-vinculin and the 150-kDa caldesmon proportion increases mainly in the postnatal period, during several months after birth. In the "Discussion," phenotypes of SMCs from developing aorta were compared to those from different layers of the adult aortic wall.     

The expression and functional activities of smooth muscle myosin and non‐muscle myosin isoforms in rat prostate

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC_17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC_17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains

Two distinct cDNA clones for nonmuscle myosin heavy chain (MHC) were isolated from a chicken fibroblast cDNA library by cross-hydridization under a moderate stringency with chicken gizzard smooth muscle MHC cDNA. These two fibroblast MHC and the gizzard MHC are each encoded in different genes in the chicken genome. Northern blot analysis showed that both of the nonmuscle MHC mRNAs were expressed not only in fibroblasts but also in a variety of tissues including brain, lung, kidney, spleen, and skeletal, cardiac and smooth muscles. However, the relative contents of the two nonmuscle MHC mRNAs varied greatly among tissues. The encoded amino acid sequences of the nonmuscle MHCs were highly similar to each other (81% identity) and to the smooth muscle MHC (81-84%), but much less similar to vertebrate skeletal muscle MHCs (38-41%) or to protista nonmuscle MHCs (35-36%). A phylogenic tree of MHC isoforms was constructed by calculating the similarity scores between these MHC sequences. An examination of the tree showed that the vertebrate sarcomeric (skeletal and cardiac) MHC isoforms are encoded in a very closely related multigene family, and that the vertebrate non-sarcomeric (smooth muscle and nonmuscle) MHC isoforms define a distinct, less conserved MHC gene family.     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate