Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M.
We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1. This new isoform contains an insert in the 542-597 position not present in the M110 previously cloned (Chen et al. (1994) FEBS Lett. 356, 51-55) from the same species. The encoded cDNA was expressed as a soluble GST-fusion protein in E. coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle. In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 +/- 3.5-fold (S.E. of the mean, n = 3). In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 microM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 microM) to relax the muscle. These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity. The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.
Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
C M
CM
Haystead
P
P
Gailly
A P
AP
Somlyo
A V
AV
Somlyo
T A
TA
Haystead
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
D018076:DNA, Complementary; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase