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• W2017507984 abstract "Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala → Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation. Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala → Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation. protein kinase C nonmuscle myosin heavy chain calcium/calmodulin-dependent protein kinase phorbol 12-myristate 13-acetate 2-[1-(3-dimethylaminopropyl)1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine phosphate N-(2-[N-{4-chlorocinnamyl]-N-methylaminomethyl]phenyl)-N-(2-hydroxyethyl)-4-methoxybenezenesulfonamide antibody to human NMHC-IIA carboxyl terminus extracellular regulated kinase N-tosyl-l-lysine chloromethyl ketone polyacrylamide gel electrophoresis green fluorescent protein hydrophobic non-basic 4-morpholineethanesulfonic acid dimethylsphingosine bovine serum albumin dinitrophenol Dulbecco's modified Eagle's medium Stimulation of sensitized mast cells by antigen leads to the activation of signal transduction pathways including signals generated through the mobilization of calcium and PKC1 (1Katakami Y. Kaibuchi K. Sawamura M. Takai Y. Nishizuka Y. Biochem. Cell Biol. 1984; 121: 573-578Google Scholar, 2Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 3Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar). One of the responses to these signals is the fusion of secretory granules with the plasma membrane, allowing release of preformed mediators such as histamine and serotonin from the cell. Calcium and PKC are necessary and sufficient for secretion in permeabilized cells (4Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar). Fusion of the secretory granules with the plasma membrane is accompanied by cytoskeletal rearrangements, which may facilitate fusion by removing a barrier of actomyosin which physically separates the two entities. Previous work has demonstrated that activation of the rat mast cell line RBL-2H3 with antigen or with calcium ionophore leads to the rapid PKC-dependent serine phosphorylation of nonmuscle myosin on both light chains and heavy chains (5Ludowyke R.I. Peleg I. Beaven M.A. Adelstein R.S. J. Biol. Chem. 1989; 264: 12492-12501Abstract Full Text PDF PubMed Google Scholar). The phosphorylation of nonmuscle myosin heavy chain (NMHC)-II, which occurs close to the carboxyl terminus at the end of the coiled-coil region, has been proposed to contribute to rearrangement of the actomyosin cytoskeleton and thus facilitate secretory granule fusion at the plasma membrane and resultant mediator release (5Ludowyke R.I. Peleg I. Beaven M.A. Adelstein R.S. J. Biol. Chem. 1989; 264: 12492-12501Abstract Full Text PDF PubMed Google Scholar).Recent studies in a variety of cell lines have demonstrated that NMHC-II can also be threonine phosphorylated in response to stimuli. In rat PC12 cells, NMHC-II phosphorylation in response to bradykinin was shown to be calcium-dependent, and was regulated by activity of the small GTPase Rac. NMHC-II phosphorylation was accompanied by loss of cortical myosin (6van Leeuwen F.N. van Delft S. Kain H.E. van der Kammen R.A. Collard J.G. Nat Cell Biol. 1999; 1: 242-248Crossref PubMed Scopus (183) Google Scholar). Calcium-dependent phosphorylation of NMHC-II was also shown in response to nutrient stimulation in the rat insulinoma cell line RINm5F, and was proposed to play a role in insulin secretion (7Wilson J.R. Biden T.J. Ludowyke R.I. Diabetes. 1999; 48: 2383-2389Crossref PubMed Scopus (22) Google Scholar). However, in neither study was the site of threonine phosphorylation of the myosin heavy chain determined. In light of these findings, it was of interest to determine whether threonine phosphorylation of NMHC-II also occurs in mast cells. Here we demonstrate that stimulation of RBL-2H3 m1 cells, an RBL-2H3 cell line made to express the muscarinic m1 receptor (8Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Crossref PubMed Scopus (58) Google Scholar), with antigen or carbachol leads to calcium-dependent threonine phosphorylation of NMHC-IIA. We provide evidence that Thr-1940, situated distal to the coiled-coil region within a CaM kinase consensus sequence, is the residue phosphorylated. We also demonstrate that CaM kinase II is activated by antigen and carbachol with time courses similar to those obtained for NMHC-IIA threonine phoshorylation. Finally, a specific inhibitor of CaM kinase attenuates threonine phosphorylation of NMHC-IIA, and also inhibits secretion in response to carbachol.MATERIALS AND METHODSRBL-2H3 m1 cells and Y79 cells were generously provided by Drs. Michael Beaven and Sachiyo Kawamoto (NHLBI), respectively, and were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. PMA, GF109203X, BAPTA-AM, dimethylsphingosine, autocamtide-2, KN-92, and KN-93 were obtained from Calbiochem (San Diego CA). Carbachol, BSA-DNP conjugate and phosphoamino acids were obtained from Sigma. Mouse anti-dinitrophenol IgE was obtained from M. Beaven or from Sigma. Anti-phosphothreonine antibody (rabbit polyclonal) was obtained from Zymed Laboratories Inc.(South San Francisco CA). Anti-phospho-ERK and protein A/G-agarose were from Santa Cruz Biotech (Santa Cruz CA). Chymotrypsin-TLCK was from Worthington Biochemicals (Lakewood, NJ). CaM kinase II was from New England Biolabs (Beverly, MA). Rabbit polyclonal antibodies to platelet myosin, which recognizes epitopes primarily in the rod region of NMHC-IIA, and to the carboxyl terminus of human NMHC-IIA (anti-HA), have been described (9Phillips C.L. Yamakawa K. Adelstein R.S. J. Muscle Res. Cell Motil. 1995; 16: 379-389Crossref PubMed Scopus (107) Google Scholar). A rabbit polyclonal antibody raised against 13 amino acid residues at the amino terminus of human NMHC-IIA was kindly provided by Dr. Mary Anne Conti (NHLBI). Anti-GFP was obtained fromCLONTECH (Palo Alto, CA). 4–20% SDS-polyacrylamide gels were obtained from FMC (Rockland, ME), while 4–12% NuPAGE gels were obtained from Novex (San Diego, CA). A synthetic peptide corresponding to amino acids 1933–1951 of rat NMHC-IIA was obtained from Research Genetics (Huntsville, AL).Antigen Stimulation of Mast CellsRBL-2H3 cells were incubated overnight with anti-dinitrophenol IgE (0.5 μg/ml), washed with phosphate-buffered saline, and the medium replaced with DMEM containing 0.1% BSA. Cells were then stimulated with DNP-BSA (20 ng/ml), washed twice with ice-cold phosphate-buffered saline containing 5 mm EDTA and 1 mm sodium vanadate, lysis buffer added, and the cells scraped from the plate with a plastic scraper. Lysis buffer consisted of 25 mm Hepes, pH 7.5, 0.3m NaCl, 1.5 mm MgCl2, 0.1% Triton X-100, 1 mm sodium orthovanadate, 20 mmβ-glycerophosphate, 0.2 mm EDTA, 0.5 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 μm leupeptin, 0.15 units/ml aprotinin at 4 °C. Cell lysates were transferred to a microcentrifuge tube, and Triton-insoluble material pelleted at 14,000 × g for 10 min. After taking an aliquot for protein determination (10Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) to permit equal protein loading of gel lanes, samples were heated in sample buffer and subjected to SDS-PAGE. Samples were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore) and subjected to immunoblotting using standard methods. Bound secondary antibody was detected using luminol blotting reagents (Santa Cruz Biotech), and the signal captured on Biomax MR film (Kodak). Films were scanned using a laser densitometer (Molecular Dynamics) and bands quantified using ImageQuant software.ImmunoprecipitationCleared cell lysates prepared as above were rocked at 4 °C with the appropriate antibody for 1 h. Protein A/G-agarose was then added, and rocking continued for a further 2 h. The protein A/G-agarose beads were washed 4 times with ice-cold phosphate-buffered saline, and then incubated with sample buffer at 75 °C for 10 min to remove bound protein from the beads. The supernatant was subjected to SDS-PAGE and immunoblotting as described above.Proteolytic Localization of the Threonine Phosphorylation SiteFor chymotryptic digestion, cell lysate (2 mg/ml) was incubated with TLCK-treated chymotrypsin, 20 μg/ml at 25 °C. Aliquots were taken at time points, heated with sample buffer, and subjected to SDS-PAGE and immunoblotting. For hydroxylamine cleavage, cell lysate was diluted 1:10 with 2 m hydroxylamine hydrochloride, 6 m guanidine hydrochloride which had been adjusted to pH 9.0 with LiOH. The mixture was incubated at 45 °C for 3 h, dialyzed against 6 m urea, 40 mm Hepes, pH 7.5, and concentrated by centrifugation in a Microcon 10 centrifugal filter (Millipore). An aliquot of the concentrated digested lysate was then subjected to SDS-PAGE and immunoblotting.Fusion Protein Expression and MutationA 1906-base pair fragment (including 3′-untranslated residues), obtained from a clone of mouse NMHC-IIA provided by M. Conti, was subcloned into pGEX-5T2 to give a glutathione S-transferase fusion protein including the carboxyl-terminal 199 amino acids of NMHC-IIA. The threonine residue located 22 amino acids from the carboxyl terminus, corresponding to rat NMHC-IIA Thr-1940, was mutated to Ala using the Quikchange system (Stratagene, La Jolla, CA). The primers used were 5′-TGTTCGGAAAGGCGCCGGCGACTGCTCAG-3′ and the complementary antisense primer; the mutated residue, shown in bold, converts Thr to Ala and introduces an SfoI site for rapid clone selection. A 450-base pair fragment was subcloned from a clone encoding human NMHC-IIA (Dr. Qize Wei, NHLBI) into pGEX-4T1 to give a glutathioneS-transferase fusion protein encoding the carboxyl-terminal 150 amino acids of NMHC-IIA. Ala-1940 was mutated to Thr using the primers 5′-CCGGAAAGGCACCGGGATGGCTCC-3′ and the complementary antisense primer; the mutated residue, shown in bold, converts Ala to Thr and removes an SfoI site. SDS-PAGE-purified primers were obtained from Life Technologies, Inc. (Gaithersburg MD). Fusion proteins were expressed in Escherichia coli and purified by standard methods (11Smith D.B. Johnson K.S. Gene (Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5035) Google Scholar).Mammalian Expression ExperimentsMammalian expression vector encoding constitutively active CaM kinases I, II, and IV (12Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (636) Google Scholar) were generously provided by Dr. Richard Maurer (Oregon Health Sciences University). An expression vector encoding human NMHC-IIA with GFP fused to the amino terminus was kindly provided by Q. Wei. Ala-1940 was mutated to Thr as described above for the human pGEX-4T1 construct. Transfection was performed using Effectene (Qiagen, Valencia, CA) following the manufacturer's standard protocol. pAdVantage (Promega, Madison, WI) was co-transfected with the expression vectors to improve expression levels by inhibiting dsRNA-activated inhibitor.In Vitro Phosphorylation with CaM Kinase II and CaM Kinase AssayProtein or peptide substrates were incubated with CaM kinase II, and where indicated [γ-32P]ATP, 250 μCi/mmol, in a reaction buffer consisting of 20 mm Tris, pH 7.5, 10 mm MgCl2, 2 mmCaCl2, 2.4 mm calmodulin, 0.5 mmdithiothreitol, 0.1 mm EDTA, 50 μm ATP. Phosphorylated proteins and peptides were then separated on NuPAGE 4–12% gels run in MES buffer. CaM kinase II activity in cell lysates was determined by phosphorylation of autocamtide-2 and capture of the phosphorylated peptide on Whatman P81 phosphocellulose as described previously (13Miralem T. Templeton D.M. Biochem. J. 1998; 330: 651-657Crossref PubMed Scopus (25) Google Scholar).Phosphoamino Acid DeterminationAfter in vitrolabeling of peptide, the reaction mixture was lyophilized and taken up in 6 m HCl, 50 μl. The mixture was incubated at 110 °C for 1 h, and lyophilized after addition of 400 μl of water. The mixture was dissolved in electrophoresis buffer (glacial acetic acid/pyridine/water, 10:1:189, pH 3.5) (14van der Geer P. Hunter T. Electrophoresis. 1994; 15: 544-554Crossref PubMed Scopus (125) Google Scholar), marker phosphoamino acids added, and an aliquot spotted on to a 20 × 20-cm glass-backed cellulose TLC plate. Electrophoresis was carried out for 30 min at 1.3 kV, the plate dried, and the phosphoamino acids visualized with ninhydrin. The plate was then subjected to autoradiography.Measurement of Secretion by Hexosaminidase AssayThe release of the secretory granule marker hexosaminidase was measured as described previously (15Choi O.H. Lee J.H. Kassessinoff T. Cunha-Melo J.R. Jones S.V. Beaven M.A. J. Immunol. 1993; 151: 5586-5595PubMed Google Scholar). Released hexosaminidase was expressed as a percentage of total hexosaminidase.Statistical AnalysisResults are presented as mean ± S.E. Comparisons between 2 groups were performed by paired or unpaired Student's t test as appropriate. Comparisons between more than 2 groups were made by repeated measure analysis of variance followed by post-hoc Tukey HSD test to assess significance of differences between individual groups.DISCUSSIONThe activation of secretory cells and release of mediators from secretory granules requires the granules to penetrate a cortical cytoskeletal barrier of actomyosin and other components in order to fuse with the plasma membrane and release their contents into the extracellular space (23Burgoyne R.D. Cheek T.R. Biosci. Rep. 1987; 7: 281-288Crossref PubMed Scopus (120) Google Scholar). Previous work has shown that stimulation of sensitized mast cells with antigen leads to the rapid PKC-dependent phosphorylation of nonmuscle myosin on serine residues in both the light and heavy chains (5Ludowyke R.I. Peleg I. Beaven M.A. Adelstein R.S. J. Biol. Chem. 1989; 264: 12492-12501Abstract Full Text PDF PubMed Google Scholar). The kinetics of early shape change and formation of myosin-deficient lamellopodia in RBL-2H3 cells correlate temporally, consistent with a role for myosin phosphorylation in the disruption of the cortical actomyosin rim that occurs during mast cell activation (24Spudich A. Cell Motil. Cytoskeleton. 1994; 29: 345-353Crossref PubMed Scopus (19) Google Scholar). This disruption may in turn facilitate interaction between the secretory granule and the plasma membrane. Here we demonstrate for the first time that CaM kinase II is activated in RBL-2H3 cells by stimuli which increase cytoplasmic calcium. The activation of CaM kinase II results in the phosphorylation of NMHC-IIA at Thr-1940, close to the carboxyl terminus and distal to the coiled-coil region.The essential role of increases in intracellular calcium and PKC activity in mast cell activation have been recognized for many years (1Katakami Y. Kaibuchi K. Sawamura M. Takai Y. Nishizuka Y. Biochem. Cell Biol. 1984; 121: 573-578Google Scholar, 2Beaven M.A. Guthrie D.F. Moore J.P. Smith G.A. Hesketh T.R. Metcalfe J.C. J. Cell Biol. 1987; 105: 1129-1136Crossref PubMed Scopus (69) Google Scholar, 3Sagi-Eisenberg R. Lieman H. Pecht I. Nature. 1985; 313: 59-60Crossref PubMed Scopus (116) Google Scholar, 4Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar). However, the potential role of the other main calcium-dependent kinase family, the CaM kinases, in mast cell activation has not been studied in depth. The CaM kinases are a family of three kinases, CaM kinases I, II, and IV. CaM kinases I and IV are monomeric, while CaM kinase II is a large multimer. CaM kinase I and IV, but not CaM kinase II, are regulated by an upstream kinase, CaM kinase kinase (25Soderling T.R. Trends Biochem. Sci. 1999; 24: 232-236Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). CaM kinase II is activated in RBL-2H3 m1 cells stimulated with antigen or with carbachol, and activation of CaM kinase II is accompanied by the threonine phosphorylation of NMHC-IIA with a similar time course. The phosphorylation is calcium-dependent, requiring both an elevation of intracellular calcium, blocked by BAPTA, and influx of extracellular calcium, blocked by brief incubation with EGTA. Carbachol stimulation led to a larger increase in NMHC-IIA phosphothreonine than antigen, paralleling the larger increase in intracellular calcium (16Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 380: 634-636Crossref PubMed Scopus (383) Google Scholar) and the greater extent of secretion (8Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Crossref PubMed Scopus (58) Google Scholar) found with carbachol relative to antigen. Phosphorylation appears to be mediated via a CaM kinase-dependent pathway rather than by PKC, since threonine phosphorylation of NMHC is inhibited by the CaM kinase inhibitor KN-93 but not by the PKC inhibitor GF109203X. In addition, treatment of the cells with PMA did not result in threonine phosphorylation of NMHC-IIA. KN-93 inhibits CaM kinase II with an IC50 of 0.37 μm (19Sumi M. Kiuchi K. Ishikawa T. Ishii A. Hagiwara M. Nagatsu T. Hidaka H. Biochem. Cell Biol. 1991; 181: 968-975Google Scholar), but also inhibits CaM kinase I and IV (IC50 2.7 and 50 μm,respectively) (26Patel R. Holt M. Philipova R. Moss S. Schulman H. Hidaka H. Whitaker M. J. Biol. Chem. 1999; 274: 7958-7968Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). While the time course of activation of CaM kinase II and effects of constitutively active CaM kinase II are consistent with a role for this enzyme in NMHC-IIA threonine phosphorylation, involvement of CaM kinase IV cannot be excluded. Since KN-93 is less effective against CaM kinase IV, a contribution from CaM kinase IV could explain the incomplete inhibition of NMHC-IIA threonine phosphorylation by KN-93.There is increasing evidence that converging calcium/calmodulin-dependent pathways are important for secretion. Calmodulin-dependent activation of myosin light chain kinase and phosphorylation of myosin light chain is required for calcium-induced cortical F-actin disassembly (27Sullivan R. Price L.S. Koffer A. J. Biol. Chem. 1999; 274: 38140-38146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). CaM kinase inhibitors also inhibit insulin secretion in pancreatic cells (28Easom R.A. Diabetes. 1999; 48: 675-684Crossref PubMed Scopus (104) Google Scholar) and gonadotropin-releasing hormone secretion from infundibular explants (29Waters W.W. Chen P.L. McArthur N.H. Moreno P.A. Harms P.G. Neuroendocrinology. 1998; 67: 145-152Crossref PubMed Scopus (12) Google Scholar), suggesting that there may be a more general role for CaM kinases in secretory processes. Threonine phosphorylation of NMHC-IIA is also found in pancreatic cells undergoing secretion (7Wilson J.R. Biden T.J. Ludowyke R.I. Diabetes. 1999; 48: 2383-2389Crossref PubMed Scopus (22) Google Scholar, 30An J. Zhao G. Churgay L.M. Osborne J.J. Hale J.E. Becker G.W. Gold G. Stramm L.E. Shi Y. Am. J. Physiol. 1999; 277: E862-8629PubMed Google Scholar). The effect of CaM kinase phosphorylation of Thr-1940 in NMHC-IIA on the interactions between actin and myosin (and possibly other cytoskeletal proteins) may give further insight into the secretory process. The ability of the CaM kinase inhibitor KN-93, but not the inactive analogue KN-92, to inhibit secretion in RBL-2H3 cells in response to carbachol lends further credence for a functional role for CaM kinase in mast cell secretion. The lack of effect of KN-93 on antigen-mediated secretion, and the absence of CaM kinase phosphorylation of NMHC-IIA in human cells, implies that NMHC-IIA threonine phosphorylation may have a facilitatory role rather than being essential for secretion. This may reflect redundancy in the IgE-mediated pathway, perhaps between PKC and CaM kinase-mediated NMHC-IIA phosphorylation. Alternatively, CaM kinase may be having additional upstream effects in the carbachol-mediated pathway which are responsible for the inhibition of carbachol-mediated secretion by KN-93.The non-helical carboxyl-terminal region of the myosin rod modulates the assembly of myosin filaments (31Cross R.A. Vandekerckhove J. FEBS Lett. 1986; 200: 355-360Crossref PubMed Scopus (23) Google Scholar, 32Ikebe M. Hewett T.E. Martin A.F. Chen M. Hartshorne D.J. J. Biol. Chem. 1991; 266: 7030-7036Abstract Full Text PDF PubMed Google Scholar, 33Hodge T.P. Cross R. Kendrick-Jones J. J. Cell Biol. 1992; 118: 1085-1095Crossref PubMed Scopus (73) Google Scholar), and it has been suggested that phosphorylation of the NMHC carboxyl terminus affects filament formation in an isoform-specific manner; phosphorylation by either PKC or casein kinase inhibits assembly of NMHC-IIB isoforms, but not of NMHC-IIA (21Murakami N. Chauhan V.P. Elzinga M. Biochemistry. 1998; 37: 1989-2003Crossref PubMed Scopus (82) Google Scholar). An alternative mechanism by which threonine phosphorylation of NMHC-IIA could affect filament assembly is through interactions with other proteins, and in particular with Mts 1. Mts 1 is a 9-kDa calcium-binding protein of the S100 family, which is up-regulated in cancer cells and highly motile cells (34Grigorian M. Tulchinsky E. Burrone O. Tarabykina S. Georgiev G. Lukanidin E. Electrophoresis. 1994; 15: 463-468Crossref PubMed Scopus (73) Google Scholar). Mts 1 binds to the non-helical region of NMHC and destabilizes filaments (35Ford H.L. Silver D.L. Kachar B. Sellers J.R. Zain S.B. Biochemistry. 1997; 36: 16321-16327Crossref PubMed Scopus (78) Google Scholar). Binding occurs at residues 1909–1937 of human platelet NMHC-IIA, and inhibits phosphorylation of Ser-1917 by PKC (36Kriajevska M. Tarabykina S. Bronstein I. Maitland N. Lomonosov M. Hansen K. Georgiev G. Lukanidin E. J. Biol. Chem. 1998; 273: 9852-9856Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). It will therefore be of interest to determine whether phosphorylation of NMHC-IIA at Thr-1940 has any effect on filament assembly or Mts 1 binding. Preliminary experiments have shown that Mts 1 also inhibits phosphorylation of NMHC-IIA by CaM kinase II in vitro. 3D. B. Buxton and R. S. Adelstein, unpublished results.Calcium-dependent threonine phosphorylation of NMHC-IIA has now been shown in a range of rodent cells, including PC12 rat pheochromacytoma cells and mouse NIE-115 neuroblastoma cells (6van Leeuwen F.N. van Delft S. Kain H.E. van der Kammen R.A. Collard J.G. Nat Cell Biol. 1999; 1: 242-248Crossref PubMed Scopus (183) Google Scholar), rat islets and rat RINm5F insulinoma cells (7Wilson J.R. Biden T.J. Ludowyke R.I. Diabetes. 1999; 48: 2383-2389Crossref PubMed Scopus (22) Google Scholar), in the mouse pancreatic cell line βTC6-F7 (30An J. Zhao G. Churgay L.M. Osborne J.J. Hale J.E. Becker G.W. Gold G. Stramm L.E. Shi Y. Am. J. Physiol. 1999; 277: E862-8629PubMed Google Scholar), and in mouse fibroblasts and rat RBL-2H3 mast cells. However, increasing intracellular calcium with ionomycin did not result in threonine phosphorylation of NMHC-IIA in HeLa cells or the Y79 retinoblastoma cell line, both of which are derived from humans. While the absence of some component of the signal transduction cascade leading to myosin phosphorylation cannot be excluded, a more likely explanation is a sequence difference between rodent and human NMHC-IIA, resulting in the absence of the phosphorylated threonine or targeting residues in human cells. The carboxyl terminus of mouse NMHC-IIA was phosphorylated in vitro by CaM kinase, while human NMHC-IIA carboxyl-terminal was not. Inspection of the sequences for rat and mouse NMHC-IIA distal to the coiled-coil region showed 2 conserved threonines which are absent in human NMHC-IIA; in particular, comparison of the conserved rat (17Choi O.H. Park C.S. Itoh K. Adelstein R.S. Beaven M.A. J. Muscle Res. Cell Motil. 1996; 17: 69-77Crossref PubMed Scopus (29) Google Scholar) and mouse2 sequence surrounding Thr-1940 (Ile-Val-Arg-Lys-Gly-Thr-Gly) with the optimal CaM kinase consensus sequences for CaM kinase IV (Hyd-X-Arg-X-X-Ser/Thr) and CaM kinase IIa (Hyd-X-Arg-X-NB-Ser/Thr-Hyd) (37White R.R. Kwon Y.G. Taing M. Lawrence D.S. Edelman A.M. J. Biol. Chem. 1998; 273: 3166-3172Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) show a good correspondence, while the sequence is less well conserved in human NMHC-IIA (Met-Ala-Arg-Lys-Gly-Ala-Gly) (22Saez C.G. Myers J.C. Shows T.B. Leinwand L.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1164-1168Crossref PubMed Scopus (105) Google Scholar).This high degree of sequence conservation in rat and mouse made it a good candidate as the kinase target, which was confirmed with mutants of the NMHC-IIA fusion proteins. Mutation of Thr-1940 to Ala in mouse NMHC-IIA abrogated in vitro phosphorylation, while mutation of Ala-1940 to Thr in human NMHC-IIA restored phosphorylation by CaM kinase II. A peptide corresponding to rat NMHC-IIA residues 1933–1951 was phosphorylated in vitro by CaM kinase II on Thr-1940, indicating that the effects of mutation of residue 1940 on fusion protein phosphorylation were not due to indirect conformational changes. Co-expression experiments in HeLa cells showed that constitutively active CaM kinase II also phosphorylated mutant full-length NMHC-IIA containing Thr-1940, but not wild type NMHC-IIA. Taken together these data confirm that Thr-1940 of rodent NMHC-IIA is phosphorylated by CaM kinase II, and that the absence of this site in human NMHC-IIA explains the lack of threonine phosphorylation in stimulated human cells.The absence of threonine phosphorylation in human cells is interesting, and may imply that they are able to rearrange their actomyosin cytoskeleton with only PKC-dependent serine phosphorylation of NMHC-II, in addition to serine phosphorylation of the nonmuscle myosin light chain. Conversely, human cells could have developed an alternative phosphoacceptor pathway, as has been demonstrated for the transcription factor C/EBPβ. The highly conserved threonine phosphoacceptor (Thr-217) in the transcription factor C/EBPβ required for transforming growth factor α-mediated hepatocyte proliferation in mouse, is replaced by alanine in rat, while mouse/human Ala-105 has been replaced by Ser-105 in rat (38Buck M. Poli V. van der Geer P. Chojkier M. Hunter T. Mol. Cell. 1999; 4: 1087-1092Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The two phosphoacceptors have analogous functions, since mutation of mouse Thr-217 or rat Ser-105 with Ala blocks proliferation while replacement with Asp promotes proliferation in both cases. It will be of interest to determine by peptide mapping whether any additional phosphoacceptors have evolved in human NMHC-IIA to substitute for the phosphothreonine.The activation of CaM kinase II that we have demonstrated also raises the possibility that CaM kinase could contribute to the changes in gene expression found following mast cell activation (39Gordon J.R. Burd P.R. Galli S" @default.
• W2017507984 created "2016-06-24" @default.
• W2017507984 creator A5002243330 @default.
• W2017507984 creator A5016373230 @default.
• W2017507984 date "2000-11-01" @default.
• W2017507984 modified "2023-10-09" @default.
• W2017507984 title "Calcium-dependent Threonine Phosphorylation of Nonmuscle Myosin in Stimulated RBL-2H3 Mast Cells" @default.
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