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• W1982483822 abstract "We report here the cloning and characterization of the gene encoding the 130-kDa myosin heavy chain kinase (MHCK A) from the amoeba Dictyostelium. Previous studies have shown that purified MHCK A phosphorylates threonines in the carboxyl-terminal tail portion of the Dictyostelium myosin II heavy chain and that phosphorylation of these sites is critical in regulating the assembly and disassembly of myosin II filaments in vitro and in vivo. Biochemical analysis of MHCK A, together with analysis of the primary sequence, suggests that the amino-terminal ∼ 500 amino acids form an α-helical coiled-coil domain and that residues from position ∼ 860 to the carboxyl terminus (residue 1146) form a domain with significant similarity to the β-subunit of heterotrimeric G proteins. No part of the MHCK A sequence displays significant similarity to the catalytic domain of conventional eukaryotic protein kinases. However, both native and recombinant MHCK A displayed autophosphorylation activity following renaturation from SDS gels, and MHCK A expressed in Escherichia coli phosphorylated purified Dictyostelium myosin, confirming that MHCK A is a bona fide protein kinase. Cross-linking studies demonstrated that native MHCK A is a multimer, consistent with the presence of an amino-terminal coiled-coil domain. Southern blot analysis indicates that MHCK A is encoded by a single gene that has no detectable introns. We report here the cloning and characterization of the gene encoding the 130-kDa myosin heavy chain kinase (MHCK A) from the amoeba Dictyostelium. Previous studies have shown that purified MHCK A phosphorylates threonines in the carboxyl-terminal tail portion of the Dictyostelium myosin II heavy chain and that phosphorylation of these sites is critical in regulating the assembly and disassembly of myosin II filaments in vitro and in vivo. Biochemical analysis of MHCK A, together with analysis of the primary sequence, suggests that the amino-terminal ∼ 500 amino acids form an α-helical coiled-coil domain and that residues from position ∼ 860 to the carboxyl terminus (residue 1146) form a domain with significant similarity to the β-subunit of heterotrimeric G proteins. No part of the MHCK A sequence displays significant similarity to the catalytic domain of conventional eukaryotic protein kinases. However, both native and recombinant MHCK A displayed autophosphorylation activity following renaturation from SDS gels, and MHCK A expressed in Escherichia coli phosphorylated purified Dictyostelium myosin, confirming that MHCK A is a bona fide protein kinase. Cross-linking studies demonstrated that native MHCK A is a multimer, consistent with the presence of an amino-terminal coiled-coil domain. Southern blot analysis indicates that MHCK A is encoded by a single gene that has no detectable introns. Conventional myosin (myosin II) has been implicated as having important roles in a wide array of cellular contractile events. In Dictyostelium, genetic and cellular analyses have demonstrated that myosin II is essential for cytokinesis, multicellular morphogenesis, capping of cell-surface receptors, and efficient amoeboid locomotion(1Fukui Y. De Lozanne A. Spudich J.A. J. Cell Biol. 1990; 110: 367-378Crossref PubMed Scopus (132) Google Scholar, 2Pasternak C. Spudich J.A. Elson E.L. Nature. 1989; 341: 549-551Crossref PubMed Scopus (227) Google Scholar, 3Manstein D.J. Titus M.A. De Lozanne A. Spudich J.A. EMBO J. 1989; 8: 923-932Crossref PubMed Scopus (231) Google Scholar, 4De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (763) 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 (251) Google Scholar). Although myosin II seems to play similar roles in a variety of cell types, the in vivo mechanisms regulating myosin II assembly and localization during these processes are not well understood in most systems. In the Dictyostelium system, strong evidence now indicates that myosin II heavy chain phosphorylation has a critical role in the control of myosin assembly and localization within cells. Purified myosin II from Dictyostelium can be phosphorylated by endogenous myosin heavy chain (MHC) ( 1The abbreviations used are: MHCmyosin heavy chainMHCKMHC kinasePAGEpolyacrylamide gel electrophoresiskbkilobase pair(s)TES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.) kinases on target sites near the carboxyl-terminal portion of the myosin tail. Two distinct threonine-specific Dictyostelium MHC kinases (MHCKs) have been purified to homogeneity(6Ravid S. Spudich J.A. J. Biol. Chem. 1989; 264: 15144-15150Abstract Full Text PDF PubMed Google Scholar, 7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar), and phosphorylation of purified MHC by either of these enzymes is capable of driving the disassembly of bipolar myosin filaments at physiological salt concentrations. One of these enzymes (MHCK A) has a molecular mass of 130 kDa on SDS gels and is expressed both in growth phase cells and in starved cells that have entered the Dictyostelium developmental pathway(8Medley Q.G. Lee S.F. Cates G.A. Côté G.P. J. Cell Biol. 1991; 115 (abstr.): 29aGoogle Scholar). In vitro target sites for this enzyme have been mapped to threonine residues 1823, 1833, and 2029 of Dictyostelium MHC (9Lück-Vielmetter D. Schleicher M. Grabatin B. Wippler J. Gerisch G. FEBS Lett. 1990; 269: 239-243Crossref PubMed Scopus (72) Google Scholar, 10Vaillancourt J.P. Lyons C. Côté G.P. J. Biol. Chem. 1988; 263: 10082-10087Abstract Full Text PDF PubMed Google Scholar). myosin heavy chain MHC kinase polyacrylamide gel electrophoresis kilobase pair(s) 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. The second Dictyostelium MHCK has a molecular mass of 84 kDa and is expressed only during development(11Ravid S. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5877-5881Crossref PubMed Scopus (49) Google Scholar). This enzyme has been recently cloned and, based on sequence homology, seems to be a member of the protein kinase C family(6Ravid S. Spudich J.A. J. Biol. Chem. 1989; 264: 15144-15150Abstract Full Text PDF PubMed Google Scholar, 11Ravid S. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5877-5881Crossref PubMed Scopus (49) Google Scholar). Phosphorylation of Dictyostelium myosin II by the 84-kDa MHCK promotes filament disassembly in vitro(6Ravid S. Spudich J.A. J. Biol. Chem. 1989; 264: 15144-15150Abstract Full Text PDF PubMed Google Scholar), although it is not clear whether the target sites for the 84-kDa MHCK are identical to those for MHCK A. Dictyostelium also seems to contain an MHC kinase that phosphorylates serine residues and may contain additional threonine-specific MHC kinases, but these activities have not been purified(12Maruta H. Baltes W. Dieter P. Marme D. Gerisch G. EMBO J. 1983; 2: 535-542Crossref PubMed Scopus (42) Google Scholar, 13Kuczmarski E.R. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7292-7296Crossref PubMed Scopus (168) Google Scholar). Exposure of Dictyostelium cells to chemoattractants or to agents that stimulate receptor capping results in a transient recruitment of myosin II from a soluble pool into the cytoskeleton, suggesting that regulated assembly and disassembly of myosin filaments into the cytoskeleton are important for these processes(14Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 15Berlot C.H. Devreotes P.N. Spudich J.A. J. Biol. Chem. 1987; 262: 3918-3926Abstract Full Text PDF PubMed Google Scholar, 16Carboni J.M. Condeelis J.S. J. Cell Biol. 1985; 100: 1884-1893Crossref PubMed Scopus (47) Google Scholar, 17Liu G. Newell P.C. J. Cell Sci. 1988; 90: 123-129PubMed Google Scholar). MHC phosphorylation on threonine residues has been observed in vivo concomitant with the transient relocalization of myosin II to the cytoskeleton, suggesting that MHC kinases may play a role in redirecting myosin II back to the soluble pool after chemoattractant-stimulated recruitment(15Berlot C.H. Devreotes P.N. Spudich J.A. J. Biol. Chem. 1987; 262: 3918-3926Abstract Full Text PDF PubMed Google Scholar, 18Berlot C.H. Spudich J.A. Devreotes P.N. Cell. 1985; 43: 307-314Abstract Full Text PDF PubMed Scopus (116) Google Scholar, 19Liu G. Newell P.C. J. Cell Sci. 1991; 98: 483-490PubMed Google Scholar). In previous work, the target sites for MHCK A (threonine residues 1823, 1833, and 2029 on MHC) were mutated either to alanine, to inhibit phosphorylation, or to aspartic acid, to mimic phosphorylation(14Egelhoff T.T. Lee R.J. Spudich J.A. Cell. 1993; 75: 363-371Abstract Full Text PDF PubMed Scopus (243) Google Scholar). Elimination of these MHCK A target sites resulted in gross overassembly of myosin into the cytoskeleton and caused an array of partial defects in myosin-related contractile processes. Conversion of these MHCK A target sites to aspartic acid rendered the myosin incapable of assembling functionally into the cytoskeleton. The phenotypes of the site-directed MHC mutants provide strong support for the idea that MHCK A has a central role in the control of myosin localization in vivo. Biochemical studies on purified MHCK A have shown that the kinase requires autophosphorylation for activity, but have been unable to identify physiologically relevant molecules that may serve to regulate MHCK A activity in vivo(7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar, 20Medley Q.G. Gariepy J. Côté G.P. Biochemistry. 1990; 29: 8992-8997Crossref PubMed Scopus (27) Google Scholar). To gain further insights into the mechanisms regulating myosin II phosphorylation and localization in vivo, we have isolated and characterized the gene for the 130-kDa MHCK A. The primary sequence reported here demonstrates, surprisingly, that MHCK A does not display any significant homology to the catalytic domains of conventional eukaryotic protein kinases, yet biochemical analysis of the recombinant MHCK A protein verifies the presence of intrinsic protein kinase activity. In addition, MHCK A appears to contain a coiled-coil domain that may function in oligomerization of the kinase and a carboxyl-terminal domain containing WD sequence repeats similar to β-subunits of heterotrimeric G proteins. Monoclonal antibodies were prepared by standard techniques following immunization of mice with native MHCK A, purified as described(7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar). Twenty-four hybridomas secreting antibodies against MHCK A were detected by enzyme-linked immunosorbent assay and subcloned. The antibody designated A1 was purified using protein A-Sepharose from the ascites fluid of mice injected with the corresponding hybridoma line. The other three monoclonal antibodies used in this study (A16, A21, and A22) were derived from hybridoma supernatants. For immunoblot analysis, washed Dictyostelium cells were boiled directly in SDS sample buffer and subjected to SDS-PAGE. Samples were either stained directly with Coomassie Blue or electroblotted to Immobilon-P (Millipore Corp.) and probed with monoclonal antibody. Signal was detected using a horseradish peroxidase-linked goat anti-mouse antibody and a chemiluminescence assay (Amersham Corp.). The MHCK A gene was cloned from a λ gt11 cDNA expression library made from Dictyostelium RNA (CLONETECH). Standard protocols were used for library plating, screening, and all phage manipulations(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The library was probed with a pool of two monoclonal antibodies (A16 and A21). Positive plaques were observed at ∼ 1/100 the frequency of plaques positive for a myosin heavy chain antiserum that was used as a control. The overall frequency of MHCK A-positive phage in the library was ∼ 1/10,000. Eight clones were picked for further characterization. Three clones were observed to react with only one of the four antibodies (either A1 or A16), one clone reacted with two antibodies (A1 + A22), one clone reacted with three of the antibodies (A1 + A16 + A22), and three of the isolated clones reacted with all four monoclonal antibodies. The cDNA inserts of the clones that reacted with three or less antibodies were determined to be ∼ 1.5 kb or less in size, while the inserts from clones reacting with all four antibodies were 2 kb or larger. These reactivity patterns suggested that each of the four antibodies recognized distinct epitopes and that the smaller inserts observed correspond to partial cDNA products. Inserts from two clones ( λ 3.1 and λ 1.1) that reacted with all four monoclonal antibodies were subcloned into plasmid vectors. Restriction enzyme analysis and subsequent sequence analysis indicated partial overlap between these inserts. The λ 3.1 insert spanned from nucleotides 21 to 2025 (as numbered in Fig. 2), and the λ 1.1 insert spanned from nucleotides 246 to 3465. DNA sequence analysis was performed by constructing nested deletions of each of these inserts from both ends using the Erase-a-Base system (Promega) and sequencing with the Sequenase system (U. S. Biochemical Corp.). All portions of the gene were sequenced at least once on both strands of the gene. Analysis of the compiled sequence indicated a complete 3′-end, but did not reveal an upstream start codon. Following Southern blot analysis, a plasmid library was constructed in ClaI-digested pBR328 from a size-selected portion of a ClaI digest of genomic Ax2 DNA. Colony screens were performed (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) with a 5′-probe derived from the λ 3.1 cDNA insert, allowing the isolation of a 1.5-kb ClaI genomic fragment that overlapped the cloned cDNA and contained upstream flanking sequences. Sequence analysis of this clone revealed a putative ATG start codon 21 bases beyond the 5′-end of the λ 3.1 clone. Upstream of this in-phase ATG codon, the sequence becomes extremely AT-rich (83%), a feature diagnostic for noncoding regions in the Dictyostelium genome. Sequence analysis was performed with the Wisconsin GCG package running on a VAX computer. Data base searches with the BLAST program were performed via Internet at the National Center for Biotechnology Information at the National Institutes of Health. Computer analysis of α-helical coiled-coil potential was performed using an algorithm devised by Lupas et al. ((23Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3483) Google Scholar); see also (22Cohen C. Parry D.A.D. Science. 1994; 263: 488-489Crossref PubMed Scopus (150) Google Scholar)). Agarose gel electrophoresis, blotting to nitrocellulose, and hybridization were performed using standard conditions(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Final filter washes were performed in 1 × SSC + 0.1% SDS at 65°C for 90 min. Genomic DNA was prepared as described previously(24Egelhoff T.T. Brown S.S. Manstein D.J. Spudich J.A. Mol. Cell. Biol. 1989; 9: 1965-1968Crossref PubMed Scopus (55) Google Scholar). Approximately 1 μg of genomic DNA was loaded per gel lane. Probes were made using a random primer method following the manufacturer's protocol (Boehringer Mannheim). Probe DNA fragments were isolated as restriction fragments from the cDNA. Probe A corresponds to nucleotides 122-1493 of the sequence as numbered in Fig. 2, probe B corresponds to nucleotides 1493-2025, and probe C corresponds to nucleotides 2765-3465. Purified native MHCK A (7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar) at 10 μg/ml in 5 mM TES, 0.1 M KCl, 6% glycerol, and 0.5 mM dithiothreitol, pH 7.5, was autophosphorylated for 30 min at 25°C by the addition of 0.1 mM [ γ - 32 P]ATP (0.2 Ci/mmol). Cross-linking was carried out in 0.5 M KCl by the addition of 0.5 mM bis(sulfosuccinimidyl)suberate (Pierce) for 5 min at 25°C. For renaturation of MHCK A purified from Dictyostelium, MHCK A (0.2 μg) was subjected to SDS-PAGE and renatured following incubation in 6 M guanidine HCl as described(25Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (435) Google Scholar). The gel was then incubated for 1 h at 25°C with 3 ml of 10 mM TES, pH 7.5, 2 mM MgCl2, 1 mM dithiothreitol, and 50 μM [ γ - 32 P]ATP (0.5 Ci/mmol). Radioactivity was removed by washing in 5% trichloroacetic acid, 1% sodium pyrophosphate. The gel was then stained with Coomassie Blue, dried, and exposed to x-ray film. For analysis of recombinant protein, the MHCK A gene was expressed in Escherichia coli strain BL21 from the vector pET21d (Novagen). Following induction with isopropyl-1-thio-β-D-galactopyranoside, cells were washed and resuspended in 5 mM imidazole, 0.5 M NaCl, and 40 mM Tris, pH 7.9, and then lysed by sonication. An inclusion body fraction was isolated by centrifugation for 15 min at top speed in a microcentrifuge. Either total bacterial protein lysates or inclusion body pellets were suspended in SDS sample buffer, heated to 100°C, and subjected to SDS-PAGE (10% gel). For all samples, an amount of material equivalent to 0.1 A600 units of original culture was loaded per lane. The “full-length” MHCK A plasmid construct expresses a fusion protein in which the first seven codons of MHCK A are replaced by four codons from the vector. The truncated MHCK A construct expresses a fusion protein with the same 4 amino-terminal vector amino acids fused to residues 634-1132 of MHCK A, followed by the polyhistidine tag from the pET21d vector. The last 14 codons of the MHCK open reading frame (residues 1133-1146) are absent in both constructs, with amino acid 1132 of MHCK A fused in phase with the polyhistidine tag of pET21d. SDS-PAGE, transfer of proteins to nitrocellulose, and denaturation/renaturation were done as described (26Celenza J.L. Carlson M. Science. 1986; 233: 1175-1180Crossref PubMed Scopus (512) Google Scholar). Phosphorylation assays were performed by incubating the filter for 30 min in 30 mM Tris, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, and 0.08 μM [ γ - 32 P]ATP (3000 Ci/mmol; Amersham Corp.) at 23°C. Following the phosphorylation step, the filter was washed in Tris-buffered saline; then with 7 M guanidine, 50 mM Tris, pH 7.5, 2 mM EDTA, and 0.25% milk for 2 h; and then with 2 N HCl for 2 h, followed by a brief Tris-buffered saline rinse before being exposed to film. E. coli cells (BL21) expressing either the truncated MHCK A (residues 634-1132) or the full-length MHCK A construct were grown at 22°C for 7 h in LB medium containing 50 μg/ml ampicillin and 1 mM isopropyl-1-thio-β-D-galactopyranoside. Cells were harvested by centrifugation; washed once in 10 mM Tris, pH 7.5, 1 mM EDTA; and then resuspended in 50 mM Tris, pH 7.5, 50 mM NaCl, 50 mM EDTA, 2 mM dithiothreitol, 10 μg/ml pepstatin, and 10 μg/ml phenylmethylsulfonyl fluoride. Cells were then lysed by sonication, and a pellet was obtained by centrifugation in a microcentrifuge. The pellet was solubilized in 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, and 1.5% Sarkosyl(27Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (833) Google Scholar); centrifuged to remove insoluble material; and dialyzed overnight at 4°C against 10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. Phosphorylation reactions contained 5 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 5 μM [ γ - 32 P]ATP (50 Ci/mmol), and 0.4 mg/ml Dictyostelium myosin(7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar). Reactions were conducted at room temperature for 15 min, and samples were then subjected to SDS-PAGE and autoradiography. The monoclonal antibodies generated against MHCK A all reacted strongly with both the unphosphorylated and autophosphorylated forms of the purified MHCK, which have SDS-PAGE mobilities of 130 and 140 kDa, respectively(28Medley Q.G. Lee S.F. Côté G.P. Protein Expression Purif. 1990; 1: 155-158Crossref PubMed Scopus (2) Google Scholar). These antibodies specifically detect bands of 130 and 140 kDa when used to probe total Dictyostelium protein extracts (Fig. 1), which presumably represent the unphosphorylated and autophosphorylated forms of MHCK A in vivo. Four of the monoclonal antibodies were used to isolate the MHCK A gene from a Dictyostelium λ gt11 cDNA expression library as described under “Materials and Methods.” The complete sequence contains a single open reading frame of 1146 codons, encoding a protein with a predicted size of 128.9 kDa (Fig. 2). This closely matches the observed size of native MHCK A (130 kDa by SDS-PAGE). Amino acid sequence analysis of the MHCK A protein isolated from Dictyostelium indicated that the amino terminus of native MHCK A was blocked. Sequencing was therefore performed on purified tryptic peptide fragments of native MHCK A to confirm the identity of the cDNA clone. Five independent peptide sequences were obtained, and all were found to match the predicted translation product of the MHCK A gene (underlineditalic residues in Fig. 2). The MHCK A sequence was used to search the data base of known sequences using the BLAST algorithm. Weak but significant homologies were found in the amino-terminal portion of MHCK A (residues ∼ 100-500) to a variety of α-helical coiled-coil proteins, including myosin II tail domains, tropomyosin, paramyosins, and intermediate filament proteins. The carboxyl-terminal portion of MHCK A (residues ∼ 880-1146) displayed strong similarity to members of the “WD repeat” family of proteins (also known as “WD40” or “-transducin-like proteins”)(29Peitsch M.C. Borner C. Tschopp J. Trends Biochem. Sci. 1993; 18: 292-293Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 30Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1299) Google Scholar). Both coiled coils and WD motifs have repetitive character, so the MHCK A sequence was analyzed with the Dotplot program of the Wisconsin GCG package to identify regions with repetitive character (Fig. 3). This analysis revealed an amino-terminal region (residues ∼ 100-500) with weak repetitive character that corresponds to the portion of MHCK A bearing similarity to known coiled-coil proteins. The central portion of the MHCK A protein displayed no significant repetitive character (residues ∼ 500-880), while the carboxyl-terminal portion (residues ∼ 880-1146) displayed a distinct 7-fold repeat of the WD or β-transducin-like repeat. We have tentatively designated each of these segments of MHCK A as distinct domains (Fig. 3, bottom), and analysis of each domain is discussed below. Southern blot analysis was performed with DNA probes corresponding to each of the three segments of the gene to establish a restriction site map of the genomic locus and to determine whether the MHCK A gene is single copy or a member of a homologous gene family. Fig. 4A presents the schematic domain assignment of the MHCK A open reading frame aligned with a DNA map indicating the position of the internal HindIII restriction site at nucleotide 1493 and the positions of each of the probes used for Southern blot analysis. Fig. 4B presents a map of the MHCK A locus derived from Southern blot and cDNA sequence analyses indicating the positions of HindIII (H) and XbaI (X) sites. Probes A, B, and C all hybridized to the same genomic DNA XbaI fragment of 8 kb (Fig. 4C, lanes2). In a HindIII digest (lanes1), probe A hybridized to a 5′-HindIII fragment of 11 kb, and probes B and C each hybridized to a 3′-HindIII fragment of 9 kb. In an XbaI + HindIII double digest (lanes3), probe A reacted with a 4-kb 5′-fragment, and probes B and C both reacted with a 4.5-kb 3′-fragment. These digests, as well as several other single and double digest combinations (data not shown), indicate that the MHCK A cDNA corresponds to a single gene that has no detectable introns (small introns of less than ∼ 80 base pairs might not be detected in this analysis). The MHCK A sequence was analyzed for coiled-coil character using a computer algorithm devised by Lupas et al.(23Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3483) Google Scholar) that accurately recognizes known coiled coils in proteins such as myosin as well as short coiled-coil segments such as the “leucine zippers.” The algorithm predicted that large stretches of amino acids within the amino-terminal 500 residues of MHCK A (corresponding to the region displaying weak repetitive character and weak similarity to known coiled-coil proteins) have an almost 100% chance of forming a coiled-coil structure (Fig. 5a). Proteins with coiled-coil domains are classically found to be assembled in their native state into dimers, trimers, or tetramers. Chemical cross-linking studies were therefore performed with purified MHCK A to assess whether native MHCK A has an oligomeric structure. MHCK A autophosphorylated in the presence of [ γ 32 P-]ATP was used for these experiments to allow cross-linked products to be visualized with high sensitivity by autoradiography. Parallel cross-linking of Dictyostelium myosin II was performed as a control. Prior to cross-linking, MHC migrated on SDS gels with a mobility corresponding to that of a monomer (240 kDa), while after cross-linking, it electrophoresed with a much lower mobility, presumably corresponding to the molecular mass of an MHC dimer (Fig. 5b, lanes1 and 2). A dramatic shift in mobility was also observed for MHCK A following cross-linking. Prior to cross-linking, MHCK A migrated at a molecular mass corresponding to 130 kDa (lane3), but after cross-linking, MHCK migrated with a much lower mobility (lane4), suggesting the formation of either a trimer or tetramer. These results are consistent with the formation of a coiled-coil domain by the amino-terminal portion of MHCK A. This prediction is also in agreement with earlier observations that showed that the 130-kDa MHCK A elutes from gel filtration columns with an apparent molecular mass of >700 kDa(7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar). The central portion of MHCK A (residues ∼ 500-880) is nonrepetitive in character and seems to be the most likely portion of the protein to contain the kinase catalytic functions. Surprisingly, computer data base searches using the BLAST algorithm failed to detect any homology between this region and members of the conserved family of eukaryotic protein kinases(31Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3826) Google Scholar, 32Hanks S.K. Quinn A.M. Methods Enzymol. 1991; 200: 38-62Crossref PubMed Scopus (1082) Google Scholar). The large majority of known eukaryotic protein kinases belong to this conserved family and display blocks of similarity to each other in ∼ 11 different subregions(33Taylor S.S. Knighton D.R. Zheng J. Ten Eyck L.F. Sowadski J.M. Annu. Rev. Cell Biol. 1992; 8: 429-462Crossref PubMed Scopus (305) Google Scholar). In direct examination by eye, a possible nucleotide-binding motif (GXGXXG) was observed at position 778 (subregion I of the conventional protein kinase family; (31Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3826) Google Scholar)), and candidate residues for an invariant lysine (subregion II) are present at position 795 or 808. A candidate for a conserved glutamate (subregion III) is present at position 834. Beyond this limited similarity, however, no significant similarity to known kinase domains could be identified by eye, by analysis with the BLAST data base search algorithm, or by direct line-up using Bestfit or Dotplot analysis against known protein kinases. Another MHCK A feature suggesting significant divergence from (or unrelatedness to) the conventional family of eukaryotic protein kinases is the position of the identified GXGXXG motif relative to the G β -like domain. The G β -like domain of MHCK A begins at approximately residue 880, only 100 residues past the GXGXXG sequence. This contrasts with the large majority of currently characterized eukaryotic protein kinase catalytic domains, which all contain at least 200 or more amino acids of conserved sequence with functional importance on the carboxyl-terminal side of the GXGXXG nucleotide-binding site(31Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3826) Google Scholar, 33Taylor S.S. Knighton D.R. Zheng J. Ten Eyck L.F. Sowadski J.M. Annu. Rev. Cell Biol. 1992; 8: 429-462Crossref PubMed Scopus (305) Google Scholar). Although native MHCK A from Dictyostelium has been extensively characterized biochemically(7Côté G.P. Bukiejko U. J. Biol. Chem. 1987; 262: 1065-1072Abstract Full Text PDF PubMed Google Scholar, 20Medley Q.G. Gariepy J. Côté G.P. Biochemistry. 1990; 29: 8992-8997Crossref PubMed Scopus (27) Google Scholar), additional tests were performed to ensure that the MHCK A gene does encode a protein with bona fide kinase activity. To rule out the possibility that a minor contaminant might be responsible for the protein kinase activity in preparations of MHCK A, protein kinase reactions were carried out after gel electrophoresis of purified MHCK A, removal of SDS, and renaturation of the polypeptide in the gel(25Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (435) Google Scholar)." @default.
• W1982483822 created "2016-06-24" @default.
• W1982483822 creator A5022407492 @default.
• W1982483822 creator A5032525715 @default.
• W1982483822 creator A5045063699 @default.
• W1982483822 creator A5064407039 @default.
• W1982483822 date "1995-01-01" @default.
• W1982483822 modified "2023-09-28" @default.
• W1982483822 title "Structural Analysis of Myosin Heavy Chain Kinase A from Dictyostelium" @default.
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