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

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Abstract
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 μM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 μM) 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