Selective purification of the 20,000-Da light chains of smooth muscle myosin

Hathaway, David R.; Haeberle, Joe R.

doi:10.1016/0003-2697(83)90726-1

Cited by 129 publications

(51 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Materials-Heterotrimeric recombinant bacterially expressed ␣1␤1␥1-AMPK (16), bovine heart PKA catalytic subunits (17), chicken gizzard MLCK (18), and myosin II light chains (19) were purified as described previously. Sequencing grade trypsin (Promega); cell culture medium (Invitrogen); calmodulin, oligomycin, phenylephrine, CaM-agarose, anti-full-length MLCK antibody (clone K36), and anti-rabbit and anti-mouse IgG conjugated to peroxidase (Sigma); vasopressin, angiotensin II, endothelin-1, A23187, and STO-609 (Calbiochem); anti-phospho-Thr 172 AMPK ␣-subunit and anti-phospho-Ser 19 MLC antibodies (Bioké); anti-phospho-Ser 221 ACC2 antibody (Upstate); enhanced chemiluminescence reagents (Thermo Fisher Scientific); anti-rabbit IgG conjugated to IRDye 800 (Rockland Immunochemicals, Inc., Philadelphia); and antisheep IgG conjugated to Alexa 680 (Molecular Probes) were from the indicated sources.…”

Section: Methodsmentioning

confidence: 99%

“…Proteins were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore 19 MLC, and anti-full-length smMLCK, AMPK, and eukaryotic elongation factor-2. The membranes were incubated either in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for detection and quantification with the Odyssey infrared imager (LI-COR Biosciences) or in 5% nonfat milk for detection by enhanced chemiluminescence.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase

Horman

Morel²,

Vertommen

et al. 2008

Journal of Biological Chemistry

108

131

View full text Add to dashboard Cite

Smooth muscle contraction is initiated by a rise in intracellu-The activity of smMLCK itself can be modulated by phosphorylation, which changes its V max or affinity for Ca 2ϩ /CaM. smMLCK is phosphorylated at two sites (7, 8) by cAMP-dependent protein kinase (PKA). One site (site A, corresponding to Ser 815 in chicken and Ser 992 in rabbit smMLCK and conserved in the human, mouse, and rat sequences) lies in the C-terminal CaM-binding domain. Treatment with PKA inactivates MLCK by decreasing its affinity for Ca 2ϩ /CaM (9). Similar changes in affinity have also been observed after phosphorylation by Ca 2ϩ /CaM-dependent kinase II (7) and protein kinase C (10). Moreover, smMLCK contains several consensus sequences for proline-directed kinases, and phosphorylation by MAPK increases the V max with no change in affinity for Ca 2ϩ / CaM (10, 11).

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase

Horman

Morel²,

Vertommen

et al. 2008

Journal of Biological Chemistry

108

131

View full text Add to dashboard Cite

show abstract

“…Caldesmon and myosin regulatory light chain (RLC) were purified from chicken gizzard as described (17,18). A plasmid expressing the fulllength, constitutively active mouse p21-activated kinase (PAK) as a GST fusion protein was a gift of Dr. S. Bagrodia and has been previously described (19).…”

Section: Methodsmentioning

confidence: 99%

Specific Phosphorylation of Threonine by theDictyostelium Myosin II Heavy Chain Kinase Family

Luo

Crawley

Steimle

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.

show abstract

“…Calmodulin was prepared from bull testes [24]. Myosin light chains were prepared from smooth muscle myosin according to Hathaway and Haebele [25] with modification [26].…”

Section: Methodsmentioning

confidence: 99%

Requirement of the two‐headed structure for the phosphorylation dependent regulation of smooth muscle myosin

Matsuura

Ikebe

1995

FEBS Letters

View full text Add to dashboard Cite

It is known for smooth muscle myosin that while acto-HMM ATPase activity is regulated by phosphorylation, acto-S-1 ATPase activity is not regulated. To clarify the heavy chain structure required for the regulation, smooth muscle myosin containing 7 different lengths of the S-2 portion were expressed in Sf9 insect ceils using Baculovirus expression system. Myosin containing longer than 991 residues of heavy chain formed a stable two-headed structure while myosin with shorter than 944 residues of heavy chain formed a single-headed structure, indicating that the residues Gln94S-Asp 99~ are critical for the fomation of the two-headed structure. The actin activated ATPase activity of myosin mutants having a two-headed structure was activated by phosphorylation while that of myosin mutants that failed to form the two-headed structure was completely independent of phosphorylation. These results suggest that the two-headed structure is critical for the phosphorylation-dependent regulation.

show abstract

Selective purification of the 20,000-Da light chains of smooth muscle myosin

Cited by 129 publications

References 19 publications

AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase

AMP-activated Protein Kinase Phosphorylates and Desensitizes Smooth Muscle Myosin Light Chain Kinase

Specific Phosphorylation of Threonine by theDictyostelium Myosin II Heavy Chain Kinase Family

Requirement of the two‐headed structure for the phosphorylation dependent regulation of smooth muscle myosin

Contact Info

Product

Resources

About