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• W2013791336 abstract "Myosin heavy chain kinase A (MHCK A) participates in the regulation of cytoskeletal myosin assembly inDictyostelium, driving filament disassembly via phosphorylation of sites in the myosin tail. MHCK A contains an amino-terminal coiled-coil domain, a novel central catalytic domain, and a carboxyl-terminal domain containing a 7-fold WD repeat motif. We have overexpressed MHCK A truncation constructs to clarify the roles of each of these domains. Recombinant full-length MHCK A, MHCK A lacking the predicted coiled-coil domain, and MHCK A lacking the WD repeat domain were expressed at high levels in Dictyostelium cells lacking endogenous MHCK A. Biochemical analysis of the purified proteins demonstrates that the putative coiled-coil domain is responsible for the oligomerization of the MHCK A holoenzyme. Removal of the WD repeat domain had no effect on catalytic activity toward a synthetic peptide, but did result in a 95% loss of protein kinase activity when native myosin filaments were used as the substrate. Cellular analysis confirms that the same severe loss of activity against myosin occurs in vivo when the WD repeat domain is eliminated. These results suggest that the WD repeat domain of MHCK A serves to target this enzyme to its physiological substrate. Myosin heavy chain kinase A (MHCK A) participates in the regulation of cytoskeletal myosin assembly inDictyostelium, driving filament disassembly via phosphorylation of sites in the myosin tail. MHCK A contains an amino-terminal coiled-coil domain, a novel central catalytic domain, and a carboxyl-terminal domain containing a 7-fold WD repeat motif. We have overexpressed MHCK A truncation constructs to clarify the roles of each of these domains. Recombinant full-length MHCK A, MHCK A lacking the predicted coiled-coil domain, and MHCK A lacking the WD repeat domain were expressed at high levels in Dictyostelium cells lacking endogenous MHCK A. Biochemical analysis of the purified proteins demonstrates that the putative coiled-coil domain is responsible for the oligomerization of the MHCK A holoenzyme. Removal of the WD repeat domain had no effect on catalytic activity toward a synthetic peptide, but did result in a 95% loss of protein kinase activity when native myosin filaments were used as the substrate. Cellular analysis confirms that the same severe loss of activity against myosin occurs in vivo when the WD repeat domain is eliminated. These results suggest that the WD repeat domain of MHCK A serves to target this enzyme to its physiological substrate. Conventional myosin (myosin II) is involved in a wide range of contractile events in eukaryotic cells. In Dictyostelium, genetic and cellular analyses have demonstrated roles for myosin in the maintenance of cortical tension, cytokinesis, morphogenesis, capping of receptors, and cell locomotion (1De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (752) Google Scholar, 2Fukui Y. De Lozanne A. Spudich J.A. J. Cell Biol. 1990; 110: 367-378Crossref PubMed Scopus (130) Google Scholar, 3Jay P.Y. Pham P.A. Wong S.A. Elson E.L. J. Cell Sci. 1995; 108: 387-393Crossref PubMed Google Scholar, 4Pasternak C. Spudich J.A. Elson E.L. Nature. 1989; 341: 549-551Crossref PubMed Scopus (225) Google Scholar, 5Wessels D. Soll D.R. Knecht D. Loomis W.F. De Lozanne A. Spudich J. Dev. Biol. 1988; 128: 164-177Crossref PubMed Scopus (247) Google Scholar). Although the roles of myosin II appear similar in many cell types, the in vivo mechanisms regulating myosin II assembly and activity in nonmuscle cells are not well understood.Assembly of Dictyostelium myosin II bipolar filaments can be regulated by phosphorylation on the myosin II heavy chain (MHC). 1The abbreviations used are: MHC, myosin II heavy chain; MHCK, MHC kinase; MES, 4-morpholineethanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; DTT, dithiothreitol. 1The abbreviations used are: MHC, myosin II heavy chain; MHCK, MHC kinase; MES, 4-morpholineethanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; DTT, dithiothreitol. Two distinct MHC kinases (MHCKs) have been purified and cloned from Dictyostelium. A 130-kDa MHCK (MHCK A), discussed below, is expressed during both growth and development. An 84-kDa MHCK that is expressed only during development appears to contain two distinct catalytic domains, one related to protein kinase C (6Ravid S. Spudich J.A. J. Biol. Chem. 1989; 264: 15144-15150Abstract Full Text PDF PubMed Google Scholar, 7Ravid S. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5877-5881Crossref PubMed Scopus (49) Google Scholar, 8Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar) and one related to diacylglycerol kinases (9Thanos C.D. Bowie J.U. Protein Sci. 1996; 5: 782-785Crossref PubMed Scopus (14) Google Scholar). Both MHCKs are capable of phosphorylating threonine residues on the myosin tail and can drive myosin bipolar filament disassembly in vitro. A third kinase, related to MHCK A, has recently been cloned from Dictyostelium (10Clancy C.E. Mendoza M.G. Naismith T.V. Kolman M.F. Egelhoff T.T. J. Biol. Chem. 1997; 272: 11812-11815Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), but is has not yet been established whether this protein regulates myosin assembly in vivo. Thein vitro target sites of MHCK A have been mapped to threonines 1823, 1833, and 2029 in the tail region of the MHC (11Luck-Vielmetter D. Schleicher M. Grabatin B. Wippler J. Gerisch G. FEBS Lett. 1990; 269: 239-243Crossref PubMed Scopus (72) Google Scholar, 12Vaillancourt J.P. Lyons C. Côté G.P. J. Biol. Chem. 1988; 263: 10082-10087Abstract Full Text PDF PubMed Google Scholar). The physiological significance of these sites was demonstrated by mutating the sites either to alanine (3X ALA myosin) to create a nonphosphorylatable MHC or to aspartic acid (3X ASP myosin) to mimic phosphorylated MHC (13Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar). In vivo, cells expressing 3X ALA myosin display severe myosin II overassembly in the cytoskeleton, whereas cells expressing 3X ASP myosin display severely reduced myosin II assembly in the cytoskeleton, resulting in a block in cytokinesis and development. The 3X ASP cells are also unable to complete development. In all tested assays, the 3X ASP cells are phenotypically identical to MHC null cells (mhc −). These studies indicate that the MHCK A target sites play a critical role in regulated myosin II assembly in vitro and that filament assembly is required for myosin function in vivo.Subsequent cellular analysis of Dictyostelium MHCK A null cells (mhck A −) and overexpressing cell lines (MHCK A+) indicated that MHCK A regulates the cytoskeletal myosin II assembly level during both growth and development (14Kolman M.F. Futey L.M. Egelhoff T.T. J. Cell Biol. 1996; 132: 101-109Crossref PubMed Scopus (56) Google Scholar). Themhck A − cells are viable, but display partial overassembly of myosin II in the cytoskeleton. MHCK A+cells display reduced myosin II assembly in the cytoskeleton and reduced efficiency of growth in suspension and fail to complete development. The MHCK A+ cell phenotype resembles those of the mhc − cells and the 3X ASP cell lines, indicating that overexpressed MHCK A can drive myosin filament disassembly in vivo.Molecular analysis of the MHCK A sequence (15Futey L.M. Medley Q.G. Côté G.P. Egelhoff T.T. J. Biol. Chem. 1995; 270: 523-529Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) indicates that it has an amino-terminal domain that is predicted to have an α-helical coiled-coil structure, a catalytic domain that is not related to conventional protein kinases, and a carboxyl-terminal domain that contains the 7-fold WD repeat motif (16Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1284) Google Scholar, 17Neer E.J. Smith T.F. Cell. 1996; 84: 175-178Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) characteristic of β-subunits of heterotrimeric G proteins. MHCK A is activated 50-fold by autophosphorylation (18Medley Q.G. Gariepy J. Côté G.P. Biochemistry. 1990; 29: 8992-8997Crossref PubMed Scopus (27) Google Scholar), and autophosphorylation is increased by polyanions such as DNA, heparin, phosphatidylserine, and phosphatidylinositol (19Medley Q.G. Bagshaw W.L. Truong T. Côté G.P. Biochim. Biophys. Acta. 1992; 1175: 7-12Crossref PubMed Scopus (18) Google Scholar).It has been demonstrated recently that the MHCK A catalytic domain is a prototype for a completely novel family of protein kinases unrelated to the conventional eukaryotic protein kinase superfamily (10Clancy C.E. Mendoza M.G. Naismith T.V. Kolman M.F. Egelhoff T.T. J. Biol. Chem. 1997; 272: 11812-11815Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Futey L.M. Medley Q.G. Côté G.P. Egelhoff T.T. J. Biol. Chem. 1995; 270: 523-529Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,20Côté G.P. Luo X. Murphy M.B. Egelhoff T.T. J. Biol. Chem. 1997; 272: 6846-6849Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 21Redpath N.T. Price N.T. Proud C.G. J. Biol. Chem. 1996; 271: 17547-17554Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Ryazanov A.G. Ward M.D. Mendola C.E. Pavur K.S. Dorovkov M.V. Wiedmann M. Erdjument-Bromage H. Tempst P. Parmer T.G. Prostko C.R. Germino F.J. Hait W.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4884-4889Crossref PubMed Scopus (166) Google Scholar). This novel group includes mammalian elongation factor-2 kinase, also known as calcium/calmodulin-dependent protein kinase III. Given its novel structure and catalytic domain, we have performed a structure-function dissection of MHCK A to identify the biochemical and physiological roles of the domains that flank the catalytic domain. The results reported here indicate that the amino-terminal coiled-coil domain is responsible for MHCK A oligomerization and that the WD repeats are important for full activity against native myosin II.RESULTSExpression of MHCK A ConstructsA schematic representation of full-length MHCK A and constructs in which the amino-terminal coiled-coil (ΔCoil-MHCK A) or the carboxyl-terminal WD repeat (ΔWD-MHCK A) domains were truncated are shown in Fig. 1. The three MHCK A constructs were overexpressed in the Dictyostelium mhck A − cell line by fusing each cDNA segment to an actin-15 promoter in an extrachromosomal vector. Once established, transfected cell lines were maintained in a 50 μg/ml concentration of the selective antibiotic G418. This elevated G418 concentration resulted in significantly higher kinase expression (3–4-fold) relative to the previously described overexpression of full-length MHCK A (14Kolman M.F. Futey L.M. Egelhoff T.T. J. Cell Biol. 1996; 132: 101-109Crossref PubMed Scopus (56) Google Scholar). The expression of each kinase was quantified by phosphoimaging of Western blots performed with MHCK A polyclonal antibodies and 125I-labeled anti-rabbit secondary antibody using each of the three purified proteins (described below) to generate a standard curve for each corresponding cell line, including wild-type cells. Calculations were performed using this Western blot analysis to generate estimates of the in vivoexpression levels of each recombinant kinase construct. Based upon this analysis, recombinant full-length MHCK A is expressed at 27 μm, ΔCoil-MHCK A is expressed at 46 μm, and ΔWD-MHCK A is expressed at 90 μm. MHCK A expression in the parental cell line JH10 was calculated to be 0.3 μm. These expression levels represent 90- and 153-fold overexpression of recombinant full-length MHCK A and ΔCoil-MHCK A, respectively, and 300-fold overexpression of ΔWD-MHCK A relative to that of MHCK A in the parental cell line (Fig. 1). By comparison, we calculate myosin II in Dictyostelium to be ∼6 μm.CytokinesisPrevious studies have demonstrated that myosin II is essential for cytokinesis as assayed by growth of cells in suspension cultures (1De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (752) Google Scholar).mhc − cells are unable to form a contractile ring, which results in the cells becoming large and multinucleated without any increase in cell number (1De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (752) Google Scholar, 27Manstein D.J. Titus M.A. De Lozanne A. Spudich J.A. EMBO J. 1989; 8: 923-932Crossref PubMed Scopus (229) Google Scholar). Similar phenotypes have been observed in 3X ASP cells and in MHCK A+ cells (13Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar,14Kolman M.F. Futey L.M. Egelhoff T.T. J. Cell Biol. 1996; 132: 101-109Crossref PubMed Scopus (56) Google Scholar). We compared growth rates of MHCK A truncation cell lines in suspension cultures as an in vivo test of myosin function (Fig. 2 a). The mhck A − cell line, which was previously shown to grow at a similar rate compared with control cells (14Kolman M.F. Futey L.M. Egelhoff T.T. J. Cell Biol. 1996; 132: 101-109Crossref PubMed Scopus (56) Google Scholar), was used as a control (filled circles). Cells that overexpress full-length MHCK A (filled squares) and ΔCoil-MHCK A (open squares) in the mhck A − cell line were unable to grow in suspension. These MHCK A+ and ΔCoil-MHCK A+ cells increased in size, were multinucleated, and eventually lysed under suspension culture conditions. In contrast, these cells grew at relatively normal rates when maintained as attached cells in plastic Petri dishes. Interestingly, when the WD repeats of MHCK A were removed (ΔWD-MHCK A+) (open circles), these cells grew at a similar rate and final density as mhck A −cells, indicating that myosin function was not impaired during cytokinesis. The ΔWD-MHCK A+ cells also displayed normal cell size during growth in suspension (data not shown).Figure 2a, suspension growth of cells expressing MHCK A truncation constructs. Cultures inoculated at 105cells/ml were grown in suspension in HL5 with 50 μg/ml G418 (themhck A − control cell line was transfected with vector pLittle for G418 resistance) and rotated at 200 rpm.b, synchronous morphogenesis of cells expressing MHCK A truncation constructs. Cells were harvested in log phase growth from Petri dishes and plated on agar starvation plates. Pictures were taken 48 h after plating.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DevelopmentPrevious studies have shown that mhc −cells, 3X ASP cells, and MHCK A+ cells are unable to complete development (13Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 14Kolman M.F. Futey L.M. Egelhoff T.T. J. Cell Biol. 1996; 132: 101-109Crossref PubMed Scopus (56) Google Scholar, 27Manstein D.J. Titus M.A. De Lozanne A. Spudich J.A. EMBO J. 1989; 8: 923-932Crossref PubMed Scopus (229) Google Scholar). We therefore used development as anin vivo test for myosin function in the MHCK A truncation cell lines (Fig. 2 b). MHCK A+ cells and ΔCoil-MHCK A+ cells arrested at mound stage and were unable to complete development, whereas control (mhck A −) and ΔWD-MHCK A+ cells completed development and formed spores at normal rates.Overexpression of MHCK A and ΔCoil-MHCK A causes defects in myosin filament-dependent processes as assayed in vivoby cytokinesis and development, suggesting that these MHCK A constructs drive myosin filament disassembly in vivo. In contrast, no defects in cytokinesis or development were observed in the ΔWD-MHCK A cell line. Interestingly, ΔWD-MHCK A had the highest expression compared with the other MHCK A-expressing cell lines (Fig. 1), with 300-fold higher expression than wild-type cells. Lack of phenotypic defects in ΔWD-MHCK A+ cells therefore is not due to lower expression of the kinase. As reported previously (14Kolman M.F. Futey L.M. Egelhoff T.T. J. Cell Biol. 1996; 132: 101-109Crossref PubMed Scopus (56) Google Scholar), elevated MHC phosphorylation in crude cell lysates was observed in MHCK A+ cells. Similar behavior was observed in ΔCoil-MHCK A+ cells, whereas no detectable increase in MHC phosphorylation was observed in lysates of ΔWD-MHCK A+cells (data not shown).Elimination of Myosin-based Defects with 3X ALA MyosinFurther analysis was performed to confirm that the defects in cytokinesis and development in MHCK A+ and ΔCoil-MHCK A+ cell lines were due to phosphorylation of myosin II and disassembly of filaments. Cell lines were constructed in which either wild-type myosin or 3X ALA myosin was expressed in anmhc − background. The 3X ALA myosin bears mutations in the in vitro mapped target sites of MHCK A in the myosin II tail. This 3X ALA myosin is predicted to be resistant to disassembly in vivo by overexpressed MHCK A if the in vitro mapped target sites are also the physiologically relevantin vivo target sites. Clonal populations of these cells expressing either wild-type or 3X ALA MHC were transfected with cDNA encoding full-length mhck A or Δcoil-mhck A to test whether the 3X ALA myosin mutation relieves the defects induced by MHCK A or ΔCoil-MHCK A overexpression. When cDNA expressing MHCK A or ΔCoil-MHCK A was transfected into cells expressing wild-type myosin, they failed to grow in suspension (Fig.3 a, filled and open squares, respectively), as expected based upon results presented above. This defect in cytokinesis caused by overexpression of MHCK A or ΔCoil-MHCK A was eliminated in cells expressing 3X ALA myosin (Fig.3 a, filled and open triangles, respectively). Overexpression of MHCK A or ΔCoil-MHCK A in wild-type myosin cells caused the cells to become large and multinucleated in suspension culture, whereas 3X ALA myosin cell lines expressing these kinase constructs remained small and displayed no increase in size (Fig. 3 b) in suspension culture. In addition, wild-type MHC cells that overexpress either full-length MHCK A or ΔCoil-MHCK A constructs were not able to complete development and arrest at the mound stage, whereas 3X ALA MHC cells that overexpress either kinase construct were able to complete development (data not shown). These results are consistent with the hypothesis that full-length MHCK A and ΔCoil-MHCK A hyperphosphorylate myosin on residues 1823, 1833, and 2029 in vivo (or a subset of these residues), resulting in myosin disassembly and consequently an mhc −phenotype.Figure 3a, rescue of MHCK A+ or ΔCoil-MHCK A+ phenotypes with 3X ALA myosin. The cDNA encoding mhck A or Δcoil-mhck A was transfected into Dictyostelium cells expressing either wild-type MHC (WT) or nonphosphorylatable 3X ALA myosin. Cultures inoculated at 105 cells/ml were grown in suspension in HL5 with 50 μg/ml G418 and rotated at 200 rpm. b, micrographs of cells overexpressing either MHCK A or ΔCoil-MHCK A in cells containing either wild-type MHC or 3X ALA MHC. Notice that cells expressing MHCK A in a wild-type myosin II background are large and multinucleated, whereas cells expressing MHCK A in a 3X ALA myosin II background are not affected by MHCK A overexpression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Purification of Full-length MHCK A, ΔCoil-MHCK A, and ΔWD-MHCK ATo determine the activity of each construct, the proteins were purified from Dictyostelium cultures. The previously reported purification method for MHCK A (26Medley Q.G. Lee S.F. Côté G.P. Methods Enzymol. 1991; 196: 23-34Crossref PubMed Scopus (10) Google Scholar) was modified to allow purification of each MHCK A construct using four to five column steps. A summary of the purification of MHCK A constructs is found in TableI, and details on the purification of each MHCK A construct are given under “Experimental Procedures.” The purification table is presented as a guide to show relative purification during the purification process. A detailed comparison of the activities of the proteins under equivalent conditions is presented below. Fig. 4 shows purified MHCK A, ΔWD-MHCK A, and ΔCoil-MHCK A after SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (panel a) or Western blot analysis (panel b). MHCK A and ΔCoil-MHCK A ran at 130 and 73 kDa, respectively. ΔWD-MHCK A ran as a doublet at 96 and 90 kDa. This could represent autophosphorylation heterogeneity or a partial proteolytic clip. Both forms were recognized by polyclonal antisera, and both became radioactive in an autophosphorylation test performed with [γ-32P]ATP.Table IPurification of MHCK A truncations from DictyosteliumVolumeProteinSpecific activityTotal activitymlmgnmol/min/mgnmol/minMHCK A Total extract9021600.0365 Supernatant8012800.0452 DE52 supernatant806400.6384 Phosphocellulose22775385 Hydroxylapatite51845818 Sephacryl S-30023460238ΔCoil-MHCK A Total extract9020700.0362 Supernatant8015600.0347 DE52 supernatant1407420.3223 Phosphocellulose19721.6115 Hydroxylapatite5313.6112 Sephacryl S-3002294.944 Mono-S71.214.417ΔWD-MHCK A Total extract1202070 Supernatant10010000.0880 DE52 supernatant1704251.4595 Phosphocellulose431083.6388 Hydroxylapatite8216.2130 Sephacryl S-300181.3518.311 AH-Sepharose1-aAH-Sepharose, aminohexyl-Sepharose.2.50.25307.51-a AH-Sepharose, aminohexyl-Sepharose. Open table in a new tab Figure 4MHCK A truncation constructs purified fromDictyostelium. a, Coomassie Blue staining of SDS-polyacrylamide gels; b, Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MHCK A Oligomerization and Rotary ShadowingSeveral lines of evidence indicate that MHCK A is an oligomeric protein kinase, including gel filtration analysis of native protein (8Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar), cross-linking studies (15Futey L.M. Medley Q.G. Côté G.P. Egelhoff T.T. J. Biol. Chem. 1995; 270: 523-529Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and coiled-coil predictive algorithms (15Futey L.M. Medley Q.G. Côté G.P. Egelhoff T.T. J. Biol. Chem. 1995; 270: 523-529Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Gel filtration chromatography performed as the final or penultimate step in each purification allowed us to test the hypothesis that the amino-terminal domain of MHCK A (residues 1–500) is responsible for oligomerization. The gel filtration analysis of full-length MHCK A and truncation constructs is presented in Fig.5. Full-length MHCK A migrated at 130 kDa on SDS-polyacrylamide gel electrophoresis, but under native conditions, gel-filtered in the void volume with an estimated molecular mass >1000 kDa. This is consistent with the 130-kDa protein being oligomeric. Rod-shaped protein domains such as coiled-coil helices gel filter with larger apparent mass that globular protein of equivalent size, so the number of MHCK A monomers in each oligomer cannot be estimated accurately from this elution behavior.Figure 5Gel filtration analysis of native molecular masses of MHCK A truncation constructs. Purified near-homogeneous preparations of MHCK A truncation constructs were applied to Sephacryl S-300 gel permeation chromatography, and elution position was determined by peptide phosphorylation and Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Deletion of the carboxyl-terminal WD repeats (ΔWD-MHCK A) resulted in gel filtration at a slightly smaller apparent mass than native MHCK A, but still >670 kDa, which is much larger than its predicted molecular size of 96 kDa. Elimination of the amino-terminal 500 amino acids caused ΔCoil-MHCK A to gel filter with an apparent molecular mass of 80 kDa, which is consistent with the predicted mass of 73 kDa. These results indicate that the amino-terminal 500 residues of MHCK A, which are predicted to form a coiled-coil structure, are responsible for MHCK A oligomerization. The computer algorithm SCORER, created by Woolfson and Alber (28Woolfson D.N. Alber T. Protein Sci. 1995; 4: 1596-1607Crossref PubMed Scopus (196) Google Scholar), predicts whether a sequence is expected to form a two- or three-stranded coiled-coil. According to this algorithm, MHCK A is predicted to form a three-stranded coiled-coil (data not shown).Full-length MHCK A was visualized by platinum rotary shadowing (Fig.6). The majority of the rotary-shadowed images appeared globular or aggregated, as in the lower right panel of Fig.6. However, a number of images were also observed that appeared to reveal a globular domain associated with an extended rod-like structure (all other panels in Fig. 6). Measurements of these images indicated an average length for the rod-like segments of ∼50 nm. The average calculated pitch of coiled-coil helices from proteins such as myosin tails is 6.7 residues/nm. If MHCK A residues 100–500 were contained entirely in a coiled-coil structure (15Futey L.M. Medley Q.G. Côté G.P. Egelhoff T.T. J. Biol. Chem. 1995; 270: 523-529Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), a rod domain of ∼60 nm would be predicted. This value is agreement with the ∼50-nm length of the rod structures observed by rotary shadowing. The apparent globular segment of the images was always observed at only one end of the rod segment, suggesting that MHCK A complexes form via parallel oligomerization rather antiparallel oligomerization.Figure 6Full-length MHCK A prepared by rotary shadowing. Purified MHCK A was sprayed in a solution containing 20–50 μg/ml protein in 50% glycerol and plated with platinum as described previously (31Tyler J.M. Branton D. J. Ultrastruct. Res. 1980; 71: 95-102Crossref PubMed Scopus (291) Google Scholar). A composite of six micrographs shows the extended rod of MHCK A. Bar = 100 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biochemical Characterization of MHCK A ConstructsAutophosphorylationPrevious studies showed that MHCK A is activated by autophosphorylation, incorporating up to 10 mol of phosphate/mol of enzyme. Incorporation of the first 3 mol of phosphate was shown to be sufficient for maximum activation (18Medley Q.G. Gariepy J. Côté G.P. Biochemistry. 1990; 29: 8992-8997Crossref PubMed Scopus (27) Google Scholar). Autophosphorylation behavior was assessed for MHCK A truncation constructs. Full-length MHCK A, ΔCoil-MHCK A, and ΔWD-MHCK A all autophosphorylated to a similar range of 8–10 mol of phosphate/mol of enzyme (Fig. 7 a). The autophosphorylation rates of each construct were independent of concentration, suggesting that autophosphorylation is an intramolecular process (data not shown).Figure 7Protein kinase activity of recombinant MHCK A constructs (MHCK A (circles), ΔCoil-MHCK A (squares), and ΔWD-MHCK A (triangles). a, phosphate incorporation into MHCK A truncation constructs by autophosphorylation. Phosphate incorporation into MHCK A (final concentration of 200 nm) truncation constructs was performed in buffer containing 10 mm TES, 2 mmMgCl2, 1 mm DTT, and 0.5 mm ATP (1000 cpm/pmol). At the indicated times, aliquots (25 μl) were removed, and 32P incorporation was determined by phosphocellulose filter paper assay. b, phosphate incorporation into peptide MH-1 by MHCK A truncation constructs. MHCK A truncation constructs were preincubated in 0.5 mm MgATP for 20 min. Phosphorylation of peptide MH-1 was initiated by the addition of kinase (2 μg/ml) and incubation for 1 min at 25 °C. Reactions were stopped by the addition of 50 mm EDTA, and32P incorporation into peptide MH-1 was determined by phosphocellulose filter paper assay. Error bars represent S.D. for triplicate samples. c, phosphate incorporation intoDictyostelium myosin II by MHCK A truncation constructs. MHCK A truncation constructs were preincubated in 0.5 mmMgATP for 20 min. Incorporation of 32P into myosin II was initiated by the addition of kinase (2.5 μg/ml) and incubation for 4 min at 25 °C. Reactions were stopped by the addition SDS sample buffer and electrophoresed on an SDS-polyacrylamide gel. Myosin bands were cut from the gel and counted in a scintillation counter. Symbols are an average of two samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activity of MHCK A Constructs toward PeptideMHCK A has been shown to phosphorylate the myosin tail on threonines 1823, 1833, and 2029. We used a 16-residue peptide (MH-1) corresponding to the sequence around Thr-2029 in the myosin tail to test the enzymatic activity of each construct. Fig. 7 b shows phosphorylation of the peptide by purified autophosphorylated MHCK A, ΔCoil-MHCK A, and ΔWD-MHCK A. The phosphorylation of the peptide obeys Michaelis-Menten kinetics; full-length recombinant MHCK A (circles)" @default.
• W2013791336 created "2016-06-24" @default.
• W2013791336 creator A5004621884 @default.
• W2013791336 creator A5022407492 @default.
• W2013791336 date "1997-07-01" @default.
• W2013791336 modified "2023-10-14" @default.
• W2013791336 title "Dictyostelium Myosin Heavy Chain Kinase A Subdomains" @default.
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