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

1990 ◽  
Vol 110 (1) ◽  
pp. 63-70 ◽  
Author(s):  
T J O'Halloran ◽  
S Ravid ◽  
J A Spudich
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Carboxyl Terminus ◽  
Dictyostelium Myosin ◽  
Myosin Heavy Chain Kinase ◽  
Acid Domain ◽  
High Level

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

scholarly journals Crystal Structure of the  -Kinase Domain of Dictyostelium Myosin Heavy Chain Kinase A

2010 ◽  
Vol 3 (111) ◽  
pp. ra17-ra17 ◽  
Author(s):  
Q. Ye ◽  
S. W. Crawley ◽  
Y. Yang ◽  
G. P. Cote ◽  
Z. Jia
Keyword(s):  
Crystal Structure ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Kinase Domain ◽  
Dictyostelium Myosin ◽  
Myosin Heavy Chain Kinase ◽  
Kinase A

scholarly journals Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment.

1987 ◽  
Vol 105 (6) ◽  
pp. 2999-3005 ◽  
Author(s):  
A De Lozanne ◽  
C H Berlot ◽  
L A Leinwand ◽  
J A Spudich
Keyword(s):  
Escherichia Coli ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Genetically Engineered ◽  
Myosin Filament ◽  
Chain Gene ◽  
Filament Formation ◽  
Ionic Strength Dependence ◽  
Expression In Escherichia Coli ◽  
Dictyostelium Myosin

The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.

scholarly journals WD Repeat Domain of Dictyostelium Myosin Heavy Chain Kinase C Functions in both Substrate Targeting and Cellular Localization

2009 ◽  
Vol 9 (2) ◽  
pp. 344-349
Author(s):  
Atiya Franklin ◽  
Linzi Hyatt ◽  
Alyssa Chowdhury ◽  
Paul A. Steimle
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cellular Localization ◽  
Myosin Ii ◽  
Content Type ◽  
Kinase C ◽  
Dictyostelium Myosin ◽  
Repeat Domain ◽  
Myosin Heavy Chain Kinase ◽  
Wd Repeat

ABSTRACT Myosin II disassembly in Dictyostelium discoideum is regulated by three structurally related myosin heavy chain kinases (myosin II heavy chain kinase A [MHCK-A], -B, and -C). We show that the WD repeat domain of MHCK-C is unique in that it mediates both substrate targeting and subcellular localization, revealing a target for regulation that is distinct from those of the other MHCKs.

scholarly journals Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyostelium Cells

2002 ◽  
Vol 13 (12) ◽  
pp. 4333-4342 ◽  
Author(s):  
Akira Nagasaki ◽  
Go Itoh ◽  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Ii ◽  
Kinase Domain ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Daughter Cells ◽  
Cleavage Process ◽  
Interphase Cells ◽  
Myosin Heavy Chain Kinase

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, themhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

Complementation of a polyamine-deficient Escherichia coli mutant by expression of mouse ornithine decarboxylase

1987 ◽  
Vol 7 (1) ◽  
pp. 564-567
Author(s):  
M Macrae ◽  
P Coffino
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Ornithine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Coli Mutant ◽  
Terminal Amino ◽  
High Level ◽  
Carboxy Terminal ◽  
Exogenous Source

Mouse ornithine decarboxylase (ODCase) cDNA was expressed at a high level in an Escherichia coli mutant deficient in polyamine biosynthesis. The expression of mouse ornithine decarboxylase relieved the dependence of the mutant on an exogenous source of polyamines, presumably by providing putrescine, the product of the enzyme. The effect on the enzymatic activity of deletions that removed carboxy-terminal amino acids of the protein was determined.

scholarly journals Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures.

1986 ◽  
Vol 103 (6) ◽  
pp. 2153-2161 ◽  
Author(s):  
L C Cerny ◽  
E Bandman
Keyword(s):  
Monoclonal Antibody ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Contractile Activity ◽  
Pectoral Muscle ◽  
Chicken Muscle ◽  
High K ◽  
Muscle Cultures ◽  
Spontaneous Contractions

The expression of neonatal myosin heavy chain (MHC) was examined in developing embryonic chicken muscle cultures using a monoclonal antibody (2E9) that has been shown to be specific for that isoform (Bandman, E., 1985, Science (Wash. DC), 227: 780-782). After 1 wk in vitro some myotubes could be stained with the antibody, and the number of cells that reacted with 2E9 increased with time in culture. All myotubes always stained with a second monoclonal antibody that reacted with all MHC isoforms (AG19) or with a third monoclonal antibody that reacted with the embryonic but not the neonatal MHC (EB165). Quantitation by ELISA of an extract from 2-wk cultures demonstrated that the neonatal MHC represented between 10 and 15% of the total myosin. The appearance of the neonatal isoform was inhibited by switching young cultures to medium with a higher [K+] which has been shown to block spontaneous contractions of myotubes in culture. Furthermore, if mature cultures that reacted with the neonatal antibody were placed into high [K+] medium, neonatal MHC disappeared from virtually all myotubes within 3 d. The effect of high [K+] medium was reversible. When cultures maintained in high [K+] medium for 2 wk were placed in standard medium, which permitted the resumption of contractile activity, within 24 h cells began to react with the neonatal specific antibody, and by 72 h many myotubes were strongly positive. Since similar results were also obtained by inhibiting spontaneous contractions with tetrodotoxin, we suggest that the development of contractile activity is not only associated with the maturation of myotubes in culture, but may also be the signal that induces the expression of the neonatal MHC.

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