Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M

Haystead, Clare M.M.; Gailly, Philippe; Somlyo, Andrew P.; Somlyo, Avril V.; Haystead, Timothy A.J.

doi:10.1016/0014-5793(95)01318-0

Cited by 37 publications

(52 citation statements)

References 15 publications

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“…Furthermore, a different 62-amino-acid deletiodinsertion in this section is apparent by comparison of the rat aorta sequences with that of the M,,,, protein from rat kidney (Fig. 1) [9]. While it is likely that most of these variations arise by alternative splicing of the mRNA, Southern blotting of rat genomic DNA revealed the presence of two closely related genes (data not shown).…”

Section: Resultsmentioning

confidence: 79%

“…Rat uterus contains ratl and rat2 forms, which possess alternative C-terminal sequences. The dashed line above the rat sequence indicates residues deleted in rat kidney M, [9]. Underlined residues in the chicken M,,,, (Ch) are deleted in some chicken gizzard forms 15, 81.…”

Section: P R R L a S T S D I D E K E N R D S S A S S I R S G S S Y A~mentioning

confidence: 99%

“…The MI ,,, subunit has been cloned from rat aorta [5], chicken gizzard [8], and rat kidney [9] cDNA libraries. The N-terminus of the M,,, subunit contains seven ankyrin repeats (residues 39-296 of the rat aorta protein), while alternative splicing in rat uterus [5] gives rise to two different C-termini ( Fig.…”

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confidence: 99%

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Identification of the Regions on the M₁₁₀ Subunit of Protein Phosphatase 1M That Interact with the M₂₁ Subunit and with Myosin

Johnson

Cohen

Chen

et al. 1997

European Journal of Biochemistry

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We have previously isolated a form of protein phosphatase-I (PPIM) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PPlC) bound to an M-complex consisting Biochem. 239,. In this paper, we establish that PPIM accounts for nearly all the myosin phosphatase activity in myofibrils, that the M,,,, and M,, subunits are present at similar concentrations in the myofibrillar fraction, and that these subunits are entirely bound to PP1. We demonstrate that the M,, subunit does not interact with PPlC, but with the C-terminal 72 residues of the M,,,, subunit, a region which is 43% identical to residues 87-161 of the M,, subunit. A fragment of the M,, subunit, M,,-(Ml-L146), which lacks the C-terminal leucine zipper, also bound to the M,,, subunit, but two other fragments M2,-(M1-E110) and M,,-(E110-K186) did not. The MI ,,, and M,, subunits were both found to be myosin-binding proteins. The C-terminal 291 residues of the M,,,, subunit, but not the C-terminal 72 residues, bound to myosin, but the N-terminal fragments M,,,-(MI -E309) and M,,,-(MI-S477) did not. Thus, the region of the M,,,, subunit that stimulates the dephosphorylation of myosin by PPlC is distinct from the region that targets PPlM to myosin. Remarkably, each myosin dimer was capable of binding about 20 mol M,, subunit and many of the M,,-binding sites were located in the myosin rod domain. The potential significance of this observation is discussed.Keywords: protein phosphatase-1 (PPl) ; myosin ; smooth muscle ; muscle contraction.Protein phosphatase-I (PPl), one of the major serinelthreonine-specific protein phosphatases in eukaryotic cells, is regulated by targetting subunits that direct it to particular subcellular loci, modify its substrate specificity, and confer the ability to be regulated by extracellular signals (reviewed in [ 1,21). A significant proportion of the PP1 in vertebrate muscle extracts is associated with myofibrils and has enhanced activity towards the Plight chain of myosin and reduced activity towards other substrates, such as glycogen phosphorylase [3, 41. When isolated from avian (chicken gizzard) [4, 51 or mammalian (pig bladder) [6] smooth muscle, this form of PP1 (PPIM) was found to be composed of three subunits, namely the catalytic subunit of PP1 Note. The novel nucleotide sequence data published here have been deposited with the sequence data banks and are available under accession numbers S74907 and S74622.(PPIC) and two other proteins with molecular masses of 110 kDa and 21 kDa, termed the MI ,,, and M,, subunits, respectively [4, 51. The M,,,, subunit is complexed to both PPlC and the M,, subunit [4], and is the component that modulates the substrate specificity of PPlC because selective removal of the M,, subunit from PPIM does not affect the rate at which either myosin or glycogen phosphorylase are dephosphorylated 171.The MI ,,, subunit has been cloned from rat aorta [5], chicken gizzard [8], and rat kidney [9] cDNA libraries. The N-terminus of the M,,, subunit contains seven ankyrin r...

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Section: Resultsmentioning

confidence: 79%

Section: P R R L a S T S D I D E K E N R D S S A S S I R S G S S Y A~mentioning

confidence: 99%

See 1 more Smart Citation

Identification of the Regions on the M₁₁₀ Subunit of Protein Phosphatase 1M That Interact with the M₂₁ Subunit and with Myosin

Johnson

Cohen

Chen

et al. 1997

European Journal of Biochemistry

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“…Thus, certain targeting proteins may occupy the phosphorylase a binding site while leaving alternative binding modes available in which other PP-1C surface grooves could accommodate specific substrates brought into immediate proximity by binding to the targeting proteins. In this respect it is of interest that in yeast PP-1C, mutation of a residue close to the COOH-terminal groove (equivalent to Arg-75 of mammalian PP-1C) reduces binding to GAC1, a G protein homolog (41).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins

Kwon

Huang

Desdouits

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

117

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“…All three MP subunits have been cloned (5,16). Expression of MPt fragments in Escherichia coli (17), and fulllength MPt in insect cells (18) have been reported. MPc in small amounts has been expressed in E. coli (19) and in insect cells (20), and MPs has been expressed in E. coli (7,21).…”

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confidence: 99%

Recombinant Small Subunit of Smooth Muscle Myosin Light Chain Phosphatase

Langsetmo

Stafford

Mabuchi

et al. 2001

Journal of Biological Chemistry

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We expressed the small subunit of smooth muscle myosin light chain phosphatase (MPs) in Escherichia coli, and have studied its molecular properties as well as its interaction with the targeting subunit (MPt). MPs (M r ‫؍‬ 18,500) has an anomalously low electrophoretic mobility, running with an apparent M r of ϳ21,000 in sodium dodecyl sulfate-gel electrophoresis. CD spectroscopy shows that it is ϳ45% ␣-helix and undergoes a cooperative temperature-induced unfolding with a transition midpoint of 73°C. Limited proteolysis rapidly degrades MPs to a stable C-terminal fragment (M r ‫؍‬ 10,000) that retains most of the helical content. Rotary shadowing electron microscopy reveals that it is an elongated protein with two domains. Sedimentation velocity measurements show that recombinant MPt (M r ‫؍‬ 107,000), intact MPs, and the 10-kDa MPs fragment are all dimeric, and that MPs and MPt form a complex with a molar mass consistent with a 1:1 heterodimer. Sequence analysis predicts that regions in the C-terminal portions of both MPs and MPt have high probabilities for coiled coil formation. A synthetic peptide from a region of MPs encompassing residues 77-116 was found to be 100% ␣-helical, dimeric, and formed a complex with MPt with a molecular mass corresponding to a heterodimer. Based on these results, we propose that MPs is an elongated molecule with an N-terminal head and a C-terminal stalk domain. It dimerizes via a coiled coil interaction in the stalk domain, and interacts with MPt via heterodimeric coiled coil formation. Since other proteins with known regulatory function toward MP also have predicted coiled coil regions, our results suggest that these regulatory proteins target MP via the same coiled coil strand exchange mechanism with MPt.Contraction of smooth muscle is regulated by phosphorylation of myosin regulatory light chain. Proper functioning of all smooth muscles requires precise control of the extent of this phosphorylation (1, 2). This control is achieved by regulating the activities of myosin light chain kinase and the corresponding phosphatase. Whereas myosin light chain kinase and its regulation by calmodulin are well studied (3), much less was known about the phosphatase until a heterotrimeric myofibrilassociated myosin phosphatase (MP) 1 was purified from avian gizzard (4, 5) and pig bladder (6). This enzyme appears to be the major if not the only myosin phosphatase in smooth muscle (7). It is composed of three subunits with apparent molecular masses (based on SDS-PAGE mobilities) of 130, 38, and 20 kDa (designated here as MPt, MPc, and MPs respectively) (reviewed in Erdödi et al. (8), and Hartshorne et al. (9)). MPc, the catalytic subunit, was identified as the ␦-isoform of type 1 protein phosphatase (PP1C␦). The targeting subunit, MPt, has been shown to bind myosin and has been attributed the function of targeting MPc to myosin. The small subunit, MPs, is a putative regulatory subunit whose exact function is thus far unknown. Several regulatory systems have been proposed to regulate the activit...

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Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M

Cited by 37 publications

References 15 publications

Identification of the Regions on the M₁₁₀ Subunit of Protein Phosphatase 1M That Interact with the M₂₁ Subunit and with Myosin

Identification of the Regions on the M₁₁₀ Subunit of Protein Phosphatase 1M That Interact with the M₂₁ Subunit and with Myosin

Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins

Recombinant Small Subunit of Smooth Muscle Myosin Light Chain Phosphatase

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Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M

Cited by 37 publications

References 15 publications

Identification of the Regions on the M110 Subunit of Protein Phosphatase 1M That Interact with the M21 Subunit and with Myosin

Identification of the Regions on the M110 Subunit of Protein Phosphatase 1M That Interact with the M21 Subunit and with Myosin

Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins

Recombinant Small Subunit of Smooth Muscle Myosin Light Chain Phosphatase

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Product

Resources

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Identification of the Regions on the M₁₁₀ Subunit of Protein Phosphatase 1M That Interact with the M₂₁ Subunit and with Myosin

Identification of the Regions on the M₁₁₀ Subunit of Protein Phosphatase 1M That Interact with the M₂₁ Subunit and with Myosin