Identification of two phosphorylated threonines in the tail region of Dictyostelium myosin II

We have previously purified and characterized a Dictyostelium myosin II heavy chain kinase which phosphorylates threonine residues (C?té, G. P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072). The phosphorylated threonines are located within a 34-kDa fragment which can be selectively cleaved from the carboxyl terminal end of the Dictyostelium myosin II tail. Tryptic and chymotryptic digests of the 34-kDa fragment phosphorylated with the kinase have now been performed and the resulting phosphopeptides isolated and sequenced. Two phosphorylated threonine residues have been identified, corresponding to residues 1833 and 2029 in the complete amino acid sequence of the Dictyostelium myosin II heavy chain. These amino acids are 87 and 283 residues, respectively, distant from the carboxyl terminus of the Dictyostelium myosin II heavy chain and are present in sections of the tail which seem to be alpha-helical coiled coils. In contrast, the three Acanthamoeba myosin II heavy chain phosphorylation sites are located within 10 residues of each other in a small globular domain at the carboxyl terminal tip of the tail (C?té, G. P., Robinson, E. A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). This suggests that the mechanism by which heavy chain phosphorylation inhibits the actin-activated ATPase activity and filament-forming properties of the two myosins may be quite different.     

Amino acid sequence around the serine phosphorylated by casein kinase II in brain myosin heavy chain

Casein kinase II from bovine brain transfers about one mole of phosphate to a serine residue near the COOH terminus of the heavy chain of myosin isolated from bovine brain. We have purified and characterized a peptide that contains this phosphoserine. The peptide was generated by chymotryptic and thermolytic digestion and was isolated by gel filtration, Fe3+ affinity chromatography, and reverse-phase high pressure liquid chromatography. Its sequence, Leu-Glu-Leu-Ser(PO4)-Asp-Asp-Asp-Asp-Glu-Ser-Lys-Ala-Ser-(Xaa)-Ile-Asn-Glu-Thr- Gln-Pro-Pro-Gln, shows that the Ser(PO4) is in an acidic environment, as is typical for casein kinase II phosphorylation sites. The "hydrophobic repeat" typical of alpha-helical coiled-coils is absent, suggesting that the sequence is part of a non-helical "tail piece" of the heavy chain. A synthetic peptide corresponding to residues 1-9 is shown to be an effective substrate for casein kinase II.     

Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.     

Filament formation and actin-activated ATPase activity are abolished by proteolytic removal of a small peptide from the tip of the tail of the heavy chain of Acanthamoeba myosin II

Actin-activated Mg2+-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of three serine residues located at the carboxyl-terminal end of each of the two 185,000-Da heavy chains; the phosphorylated molecule has full Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity. Under controlled conditions, chymotrypsin removes a small peptide containing all three phosphorylation sites from the ends of the myosin II heavy chains producing a molecule with heavy chains of 175,000 Da and undigested light chains. The length of the myosin II tail decreased from 89 to 76 nm. Chymotrypsin-cleaved myosin II has complete Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity under standard assay conditions and binds to F-actin as well as undigested myosin II in the absence, but not in the presence, of MgATP. In the presence of MgCl2, undigested myosin II forms biopolar filaments but chymotrypsin-cleaved myosin II forms only parallel (monopolar) dimers, as assessed by analytical ultra-centrifugation and rotary shadow electron microscopy. We conclude that the short segment very near the end of the myosin II tail that contains the three phosphorylatable serines is necessary for the formation of biopolar filaments and, probably as a consequence of filament formation, for the high-affinity binding of myosin II to F-actin in the presence of ATP and the actin-activated Mg2+-ATPase activity of native myosin II. This supports our previous conclusion that actin-activated Mg2+-ATPase of native myosin II is expressed only when the enzyme is in bipolar filaments with the proper conformation as determined by the state of phosphorylation of the heavy chains.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin II by copolymerization with phosphorylated and dephosphorylated peptides derived from the carboxyl-terminal end of the heavy chain

Myosin II from Acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. The amino-terminal approximately 90 kDa of each heavy chain form a globular head that contains the ATPase site and an ATP-sensitive actin-binding site. The carboxyl-terminal approximately 80 kDa of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. Phosphorylation of 3 serine residues at the tip of the tail (at positions 11, 16, and 21 from the carboxyl terminus) inactivates the actin-activated Mg2(+)-ATPase activity of myosin II filaments. Previous work had indicated that the activity of each myosin II molecule in a filament reflects the global state of phosphorylation of the filament rather than the phosphorylation state of the molecule itself. We have now purified the approximately 28-kDa carboxyl-terminal region of the heavy chain lacking the last two phosphorylation sites, and we have shown that this peptide copolymerizes with and regulates the actin-activated Mg2(+)-ATPase activities of native dephosphorylated and phosphorylated myosin II. It can be concluded from these studies that the biologically relevant enzymatic activity of myosin II is regulated by a phosphorylation-dependent conformational change in the myosin filaments.     

Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin

Phosphorylation of the myosin heavy chains of Dictyostelium discoideum is known to be inhibited following chemotactic stimulation of the cells. Effects of dephosphorylation on the assembly of myosin and on its actin-activated ATPase activity raised the question of where the phosphorylated sites are located with respect to sites responsible for polymerization and actin binding. Using seven monoclonal antibodies the binding sites of which were mapped in the electron microscope, two phosphorylation sites, i.e., threonine residues that were phosphorylated by a kinase from D. discoideum, were localized by immunoblotting of chymotryptic fragments. Two of the antibodies bound to the terminal one fifth of the tail and recognized a phosphorylated chymotryptic fragment of 38 kd. The non-phosphorylated form and single and double phosphorylated forms of this fragment were separated by two-dimensional electrophoresis. Antibody labeling of lower mol. wt. polypeptides indicated that both phosphorylation sites were located at least 32 kd from the end of the tail. A non-phosphorylated fragment, that was insoluble at low ionic strength due to polymerization, proved to be an internal cleavage product of the tail. A segment of this fragment necessary for polymerization was mapped adjacent to the phosphorylation sites.     

Phosphorylation of threonine residues on cloned fragments of the Dictyostelium myosin heavy chain

A tail fragment of Dictyostelium discoideum myosin has been cloned and expressed as a fusion protein with the N-terminal region of MS-2 polymerase. The cloned fragment was phosphorylated with myosin heavy chain kinase II from aggregation-competent D. discoideum cells that specifically phosphorylate threonine residues on the myosin tail. Phosphopeptide maps showed the same site specificity of phosphorylation with the fusion protein as a substrate as with native myosin. An improved assay for the kinase was developed in which the fusion protein is precipitated with a monoclonal antibody that inhibits polymerization of the myosin tails without preventing their phosphorylation. Sites of phosphorylation were tentatively localized to a sequence in the C-terminal region of the heavy chain where four threonine residues are found.     

Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation

The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.     

Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation

Acanthamoeba myosin IA is a globular protein composed of a 140-kDa heavy chain and a 17-kDa light chain. It expresses high actin-activated Mg2+-ATPase activity when one serine on the heavy chain is phosphorylated. We previously showed that chymotrypsin cleaves the heavy chain into a COOH-terminal 27-kDa peptide that can bind to F-actin but has no ATPase activity and a complex containing the NH2-terminal 112-kDa peptide and the light chain. The complex also binds F-actin and has full actin-activated Mg2+-ATPase activity when the regulatory site is phosphorylated. We have now localized the ATP binding site to within 27 kDa of the NH2 terminus and the regulatory phosphorylatable serine to a 20-kDa region between 38 and 58 kDa of the NH2 terminus. Under controlled conditions, trypsin cleaves the heavy chain at two sites, 38 and 112 kDa from the NH2 terminus, producing a COOH-terminal 27-kDa peptide similar to that produced by chymotrypsin and a complex consisting of an NH2-terminal kDa peptide, a central 74-kDa peptide, and the light chain. This complex is similar to the chymotryptic complex but for the cleavage which separates the 38- and 74-kDa peptides. The tryptic complex has full (K+, EDTA)-ATPase activity (the catalytic site is functional) and normal ATP-sensitive actin-binding properties. However, the actin-activated Mg2+-ATPase activity and the F-actin-binding characteristics of the tryptic complex are no longer sensitive to phosphorylation of the regulatory serine. Therefore, cleavage between the phosphorylation site and the ATP-binding site inhibits the effects of phosphorylation on actin binding and actin-activated Mg2+-ATPase activity without abolishing the interactions between the ATP- and actin-binding sites.     

Amino acid sequence of the active site of Acanthamoeba myosin II

We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.     

Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation

The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.     

Protein kinase C phosphorylates nonmuscle myosin-II heavy chain from Drosophila but regulation of myosin function by this enzyme is not required for viability in flies

Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this regulation in a complex eukaryote, we purified the Drosophila myosin-II tail expressed in Escherichia coli and showed that it was phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using a 341 amino acid tail fragment, we show that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, we generated transgenic flies that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way.     

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 serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin 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(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.     

Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.

Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.     

Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly.

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.     

Structure-function studies on Acanthamoeba myosins IA, IB, and II

Myosins IA and IB are globular proteins with only a single, short (for myosins) heavy chain (140,000 and 125,000 daltons for IA and IB, respectively) and are unable to form bipolar filaments. The amino acid sequence of IB heavy chain shows 55% similarity to muscle myosins in the N-terminal 670 residues, which contain the active sites, and a unique 500-residue C-terminus highly enriched in proline, glycine, and alanine. The C-terminal region contains a second actin-binding site which allows myosins IA and IB to cross-link actin filaments and support contractile activity. Myosins IA and IB are regulated solely by phosphorylation of one serine on the heavy chain positioned between the catalytic site and the actin-binding site that activates ATPase. Myosin II is a more conventional myosin in composition (two heavy chains and two pairs of light chains), heavy chain sequence (globular head 45% identical to muscle myosins and a coiled-coil helical tail), and structure (bipolar filaments). The tail of myosin II is much shorter than that of other conventional myosins, and it contains a 25 amino acid sequence in which helical structure is predicted to be weak or absent. The position of this sequence corresponds to the position of a bend in the monomer. Myosin II heavy chains also have a 29-residue nonhelical tailpiece which contains three regulatory, phosphorylatable serines. Phosphorylation at the tip of the tail regulates ATPase activity in the globular head apparently through an effect on filament structure.     

Protein kinase C phosphorylates both the light chains and the head portion of the heavy chains of brain myosin

Protein kinase C phosphorylated both the 19/21-kDa regulatory light chains and heavy chains of bovine brain myosin. The major phosphorylation sites of the light chains were on their threonyl residues, while those for myosin light chain kinase were on their seryl residues. Whereas several non-muscle regular myosins have been reported to be phosphorylated by different types of protein kinases at the non-helical small segments at the tail ends of the heavy chains, the phosphorylation sites for protein kinase C were localized on the head portion of the heavy chains of brain myosin. The possible role of phosphorylation of brain myosin by protein kinase C in the regulation of motility of neural cells is discussed.     

Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain

We have determined the sequence of the sites phosphorylated by protein kinase C in the turkey gizzard smooth muscle myosin light chain. In contrast to previous work (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072), two-dimensional tryptic peptide maps of both heavy meromyosin and the isolated myosin light chain showed two major phosphopeptides, one containing phosphoserine and the other phosphothreonine. We have purified the succinylated tryptic phosphopeptides using reverse phase and DEAE high pressure liquid chromatography. The serine-containing peptide, residues 1-4 (Ac-SSKR), is the NH2-terminal peptide. The phosphorylated serine residue may be either serine 1 or serine 2. The threonine-containing peptide, residues 5-16, yielded the sequence AKAKTTKKRPQR. Analysis of the yields and radioactivity of the products from automated Edman degradation showed that threonine 9 is the phosphorylation site.     

The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.     

Association of the S-100-related calpactin I light chain with the NH2-terminal tail of the 36-kDa heavy chain

Calpactin I, a Ca2+- and phospholipid-binding cytoskeletal protein, which serves as a major substrate of protein-tyrosine kinases, was isolated from bovine intestine and lung as a species containing two 36-kDa heavy chains and two 10-kDa light chains. The heavy chain is comprised of two distinct domains which can be identified by limited proteolysis: a COOH-terminal 33-kDa core, which contains the Ca2+- and phospholipid-binding sites, and an NH2-terminal tail, which contains the major site of phosphorylation by pp60v-src. To determine the site of association of the light chain on the heavy chain, we analyzed the association states of the light chain, core, and tail by sucrose gradient centrifugation after limited chymotryptic digestion. The core was not detected in higher Mr complexes with the light chain, and the tail cosedimented with a light chain dimer. The tail, isolated from chymotryptic digests and radiolabeled with 125I, was found to form a specific complex with the light chain, but not the core. The authentic tail and a synthetic peptide corresponding to residues 1-29 of the calpactin I heavy chain were both able to specifically inhibit the reassociation between heavy and light chain, whereas a synthetic peptide corresponding to residues 15-33 was inactive. These results suggest that the tail may serve as a site of regulation by light chain or phosphorylation.