NH2-terminal structural motifs in staphylokinase required for plasminogen activation

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.     

The relationship between insulin and vanadium metabolism in insulin target tissues

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) have potent mitogenic effects on granulosa and theca cells. However, their effects on steroidogenesis by these cells is controversial, and there is limited information regarding their effects on luteal cell steroidogenesis. The present study investigated the cellular distribution of the EGF receptor (EGF-R) in the rat corpus luteum (CL) by immunocytochemical staining, and the effects of EGF and TGF-alpha on progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production in cultures of luteal cells. Using a primary antibody directed against the human EGF-R peptide, specific EGF-R staining was obtained in the CL. Both small and large luteal cells had EGF-R staining. In initial cell culture experiments, treatment of freshly isolated luteal cells with EGF or TGF-alpha (0.5-50 ng/ml) for 24 h had no effect on progesterone and 20 alpha-OH-P accumulation. Addition of LH (250 ng/ml) alone caused a 3.5-fold increase in both progestins, but co-treatment with EGF or TGF-alpha produced no further enhancement of progestin accumulation. However, when cells were seeded overnight and the attached cells were washed prior to growth factor treatment for 3 days with media change every 24 h, both EGF and TGF-alpha caused dose-dependent increases in progesterone accumulation/24 h period (up to 2-fold at 50 ng/ml growth factor) on days 1 and 2 but not day 3 of treatment. 20 alpha-OH-P accumulation was similarly stimulated (up to 2.5-fold) by EGF and TGF-alpha under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.     

Cardiovascular and sympathetic effects of proadrenomedullin NH2-terminal 20 peptide in conscious rats

Proadrenomedullin NH2-terminal 20 peptide (PAMP) and adrenomedullin (AM), which are derived from the same gene, are novel vasodilative peptides and have been shown to exhibit hypotensive action in anesthetized animals. To avoid the modification via anesthesia, we investigated the effects of intravenously administered PAMP on mean arterial pressure, heart rate (HR), and renal sympathetic nerve activity (RSNA) relative to those of AM in conscious unrestrained rats. We also examined whether the arterial baroreceptor reflex was altered with the two peptides. Intravenous injection of rat PAMP (rPAMP) (10, 20 and 50 nmol/kg) and rat AM (rAM) (0.3, 1.0 and 3.0 nmol/kg) similarly elicited dose-related hypotension accompanied by increases in HR and RSNA. However, the responses to rPAMP were less potent in magnitude and shorter in duration than those to rAM. Moreover, rAM facilitated baroreflex control, whereas rPAMP attenuated it. These findings indicate that although PAMP, as well as AM, may play an important role as a circulating hormone in the systemic circulation of conscious rats, the two peptides derived from an identical origin might have different mechanisms responsible for their cardiovascular and RSNA actions.     

The NH2-terminal region of apolipoprotein B is sufficient for lipoprotein association with glycosaminoglycans

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. The co-localization of apolipoprotein (apo) B and proteoglycans within lesions has suggested that retention is due to lipoprotein interaction with these highly electronegative glycoconjugates. Both apoB100- and apoB48-containing lipoproteins, i.e. low density lipoproteins (LDLs) and chylomicron remnants, are atherogenic. This suggests that retention is due to determinants in the initial 48% of apoB. To test this, the interaction of an apoB fragment (apoB17), and apoB48- and apoB100- containing lipoproteins with heparin, subendothelial matrix, and artery wall purified proteoglycans was studied. ApoB100-containing LDL from humans and human apoB transgenic mice and apoB48-containing LDLs from apoE knockout mice were used. Despite the lack of the carboxyl-terminal 52% of apoB, the apoB48-LDL bound to heparin-affinity gel as well as did apoB100-LDL. An NH2-terminal fragment containing 17% of full-length apoB was made using a recombinant adenovirus; apoB17 bound to heparin as well as did LDL. Monoclonal antibodies against the NH2-terminal region of apoB decreased apoB100 LDL binding to heparin, whereas antibodies against the LDL receptor-binding region did not alter LDL-heparin interaction. The role of the NH2-terminal region of apoB in LDL interaction with matrix molecules was also assessed. Media containing apoB17 decreased LDL binding to subendothelial matrix by 42%. Moreover, removal of the apoB17 by immunoprecipitation abrogated the inhibitory effect of these media. Antibodies to the NH2-terminal region decreased LDL binding to matrix and dermatan sulfate proteoglycans. Purified apoB17 effectively competed for binding of LDL to artery derived decorin and to subendothelial matrix. Thus, despite the presence of multiple basic amino acids near the LDL receptor-binding domain of LDL, the NH2-terminal region of apoB is sufficient for the interaction of lipoproteins with glycoconjugates produced by endothelial and smooth muscle cells. The presence of a proteoglycan-binding site in the NH2-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.     

The NH2-terminal half of the Tn10-specified tetracycline efflux protein TetA contains a dimerization domain

The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively). An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer. In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives. TetA, TetA alpha, and TetA beta were each fused genetically to a six-histidine carboxyl-terminal tail. The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined. An interaction between alpha domains on different polypeptides which resulted in multimerization was seen. The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein. No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations. A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented.     

The Shp-2 tyrosine phosphatase has opposite effects in mediating the activation of extracellular signal-regulated and c-Jun NH2-terminal mitogen-activated protein kinases

Shp-2 is a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains. A targeted mutant allele of the Shp-2 gene with a deletion of 65 amino acids in the NH2-terminal SH2 domain was created that leads to embryonic lethality at mid-gestation in homozygous mutant mice. To define the Shp-2 function in cell signaling, we have established mutant fibroblast cell lines, and have examined the effect of the Shp-2 mutation on extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. Insulin-like growth factor (IGF)-I-induced ERK activation was completely abolished, while ERK activity upon platelet-derived growth factor and epidermal growth factor stimulation was significantly reduced and shortened in mutant cells. Stimulation of ERK by phorbol 12-myristate 13-acetate was not affected in mutant cells, but the phorbol 12-myristate 13-acetate-induced ERK activity decayed much faster compared with that in wild-type cells. In contrast, JNK activation upon heat shock was significantly enhanced in Shp-2 mutant cells. Based on these results, we conclude that Shp-2 plays differential positive regulatory roles in various mitogenic signaling pathways leading to ERK activation, and that Shp-2 is a negative effector in JNK activation by cellular stress. This is the first evidence that a tyrosine phosphatase has opposite effects in mediating the activation of ERK and JNK MAP kinases.     

The production of the ostrich NH2-terminal POMC fragment requires cleavage at a unique signal peptidase site

The NH2-terminal fragment of ostrich proopiomelanocortin was isolated and purified following acid/acetone extraction. The amino acid sequence was deduced by automatic Edman degradation of the native as well as CNBr-, tryptic-, and S. aureus protease-derived peptides. Primary structure analysis reveals its close resemblance to other known sequences, especially to amphibian POMC. The usual Trp/Gln-Cys NH2-terminal sequence found in all other homologous sequences, is replaced here by an His-Gly-Pro-Cys sequence. In addition, the gamma-MSH sequence, contrary to salmon POMC, is present and contains three substitutions, namely a Ser, an Asn, and a Lys residue substituting the normally occurring mammalian Gly, Asp, and Arg residue, respectively. Finally, the molecular weight of this fragment as deduced from ion-spray mass spectrometry and sedimentation equilibrium centrifugation is in close agreement with the proposed structure.     

The NH2-terminal region of the sickle hemoglobin beta chain. II. Characterization of monospecific antibodies

We have previously shown that antibodies specific for hemoglobin S could be fractionated by absorption of an antiserum to hemoglobin S to Sepharose containing a synthetic oligopeptide. betaS (1-13), corresponding to the first 13 amino acid residues of the beta chain of hemoglobin S. We report here that this antibody population, anti-betaS (1-13), shows considerable restriction of heterogeneity in isoelectric focusing studies and monospecificity on velocity ultracentrifugation in the presence of hemoglobin S. The binding of various hemoglobin species to anti-betaS (1-13) was studied using a double antibody radioimmunoassay with [14C]carbamoylated hemoglobin S. Carbonmonoxy-, oxy-, met-, and cyanmethemoglobin S reacted equally with the antibody, but deoxyhemoglobin (with or without organic phosphates) reacted differently. Hemoglobin A and several hemoglobin mutants with alterations in the NH2-terminal region of the beta chain did not displace labeled hemoglobin S from anti-betaS (1-13). BETAS chains reacted with the antibody, but less well than hemoglobin S, while betaA and alpha chains, and globins did not react with the antibody. The synthetic peptide, betaS (1-13), used for fractionation, reacted with the antibody about 300-fold less efficiently than hemoglobin S. BetaS (3-13) was even less reactive, while smaller peptides which included the valine residue at position 6 displaced little of the tracer [14C]carbamoylated hemoglobin S at concentrations as high as 10(-2) M. We interpret these results to indicate that this method of immunoabsorption has produced a monospecific subfraction of antibodies which is specific for the NH2-terminal region of the beta chain of hemoglobin S in its native conformation.     

A function in apoptosis other than transactivation inherent in the NH2-terminal domain of p53

This study used a cluster analysis to examine the clinical profiles of female survivors of child sexual abuse. Eighty-five participants who presented for group therapy to deal specifically with issues related to sexual abuse completed the revised version of the Minnesota Multiphasic Personality Inventory (MMPI-2; J. N. Butcher, W. G. Dahlstrom, J. R. Graham, A. Tellegen, & B. Kaemmer, 1989) as part of an extensive assessment procedure. The cluster-analytic procedure used in this study allowed 5 subgroups within the population to emerge, supporting the idea that women who report having been sexually abused as children are not a homogeneous group. Additional analyses indicated differences on the basis of cluster membership on the MMPI-2 content scales, as well other measures of psychological distress. The treatment implications of these findings are discussed.     

The NH2-terminal region of the beta chain of sickle hemoglobin. I. Synthesis and purification of oligopeptides

Five peptides from the NH2-terminal region of the beta chain of hemoglobin S, betaS (1-13), betaS (3-13), betaS (1-8), betaS (4-10), And betaS (4-8), have been synthesized by a rapid solid phase method based on the Merrifield procedure. In addition, one peptide, betaS (3-13), has also been synthesized by the original Merrifield method. We have shown that the products of the two methods are comparable, that gel filtration is a useful method for removing truncated fragments of the desired oligopeptide, and that measurement of the efficiency of coupling at each step is an important adjunct to amino acid analysis in determining purity. Peptides of the purity achieved by these methods may be used to fractionate antibodies to the native hemoglobin S, in the characterization of antigen-binding properties of specific antibodies, and in other studies of peptide-protein interactions.     

Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation

Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.     

Conformational modulation of troponin T by configuration of the NH2-terminal variable region and functional effects

Troponin T (TnT) is an essential element in the thin filament-based regulatory system of striated muscle. Alternative mRNA splicing generates multiple TnT isoforms with primary structural differences in the NH2-terminal region. The functional significance of this hypervariable NH2-terminal domain and the developmental or muscle type-specific TnT isoforms is not fully understood. We have analyzed chicken breast muscle TnT containing a metal-binding cluster [H(E/A)EAH]4-7 (Tx) in the NH2-terminal region to demonstrate potential effects of the NH2-terminal structure on the conformation of TnT [Ogut, O., and Jin, J.-P. (1996) Biochemistry 35, 16581-16590]. Using specific antibody epitope analysis on this metal-binding TnT model, this study revealed that the binding of Zn2+ to the NH2-terminal region of chicken breast muscle TnT induces extensive conformational changes in the whole protein as demonstrated by a significant decrease in binding avidity of a polyclonal anti-TnT serum which recognizes multiple epitopes on the TnT molecule. This NH2-terminal configuration-based effect is not restricted to the metal ion interaction, whereas the binding of anti-NH2 terminus monoclonal antibodies to TnT induced similar changes. Protein-binding assays have shown that the NH2-terminal variability-induced conformational changes can alter TnT's binding affinity for tropomyosin and troponin I. The results suggest a functional modulation of TnT through the configuration of the NH2-terminal domain, and this novel mechanism may mediate the physiological significance of the TnT isoform regulation.     

Synthesis and processing in vivo of the novel mouse thyrotropin beta-presubunit that contains an NH2-terminal extension sequence

Expression of the single mouse TSH beta gene gives rise to multiple mRNAs, and we have previously shown that in vitro, one of these mRNAs gives rise to a novel TSH beta-presubunit due to initiation of translation at an in-frame start site unique to this mRNA which is up-stream of the normal start site. The novel presubunit contains a 17-amino acid NH2-terminal extension sequence compared to the normal presubunit. Although this extension sequence does not have the characteristics of a normal signal sequence, the novel TSH beta-presubunit was processed in vitro by microsomal membranes. In this study we have examined the translation product of this mRNA in intact cells and whether in vivo it gives rise to a processed secreted TSH beta-subunit that has an NH2-terminal sequence different from that of the established TSH beta-subunit. Firstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it was found that immunoprecipitable TSH dimer was secreted into the medium regardless of the mRNA used for TSH beta-subunit synthesis. However, less TSH was obtained when the TSH beta-subunit was derived from the extended TSH beta-presubunit. Secondly, when COS cells were transiently transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when the TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA encoding the extended presubunit in which the down-stream AUG had been eliminated by site-specific mutagenesis. This demonstrated that the up-stream translation start site was used in the intact cell and that secreted TSH beta-subunit was derived from the extended presubunit and not from normal presubunit resulting from translational readthrough to the down-stream AUG. When secreted TSH beta-subunits derived from the normal and extended TSH beta-presubunits were digested with endoproteinase LysC, the NH2-terminal fragments were similar in size, suggesting that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate that the longer TSH beta-presubunit is synthesized in vivo and strongly suggest that it is processed in the intact cell to give a mature secreted TSH beta-subunit indistinguishable from that derived from the normal TSH beta-presubunit.     

Antibodies to an NH2-terminal fragment of betaS globin. I. Preparation and radioimmunoassay

Polypeptide fragments corresponding to the NH2-terminal 55 amino acids of betaS and betaA globins were prepared by cyanogen bromide treatment of globin and isolated by gel filtration on Sephadex G-50. Sheep were immunized with the isolated NH2-terminal fragments, and one of these sheep produced precipitating antibodies to the NH2-terminal fragment of betaS globin. These antibodies also reacted with betaA and betaS globin and hemoglobins A and S, as shown by immunodiffusion and quantitative precipitation studies. A radioimmunoassay was developed using the radioiodinated NH2-terminal fragment as tracer, and dextran-coated charcoal for separating bound and free peptide. The radioimmunoassay was used to characterize the interaction of the antibodies and the NH2-terminal fragment of betaS globin.     

Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva

When T cells become infected by the parasite Theileria parva, they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression. T. parva was found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRzeta or CD3epsilon chains were found not to be phosphorylated and T. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest that T. parva not only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.     

Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Role of the NH2-terminal region

The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.     

Function of the NH2-terminal domain of the regulatory light chain on the regulation of smooth muscle myosin

The role of the NH2-terminal domain of the 20,000-dalton light chain on the regulatory function of smooth muscle myosin was studied by exchanging it in myosin with various mutant forms. The 10 S to 6 S conformational transition as well as the thick filament formation were significantly influenced by the deletion or substitution of the amino acid residues at the NH2-terminal side of the phosphorylation site (Ser19). Whereas the deletion of Ser1-Thr10 did not significantly affect these functions, further deletion of Lys11-Arg16 completely abolished the formation of 10 S conformation and induced thick filament formation. Among the residues in this region, Arg13 and Arg16 were most important for these functions since substitution of these residues by Glu or Ala significantly altered these functions. Similar substitutions of Lys11 and Lys12 also stabilized the 6 S conformation and thick filament formation but less effectively. While the 6 S conformation was stabilized, the deletion of NH2-terminal residues did not activate the actin-activated ATPase activity. This suggests that stabilization of the 6 S conformation is not directly coupled with activation of actomyosin ATPase activity but rather a more defined conformational change around the phosphorylation site is necessary for activation. Such a change also influences the 6 S to 10 S conformation and, therefore, the filament formation. To support this notion, substitution of Lys11 and Lys12 by Glu-Glu inhibited the phosphorylation-induced activation of actomyosin ATPase activity.