MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF A RECOMBINANT 72.5 KDAFRAGMENT 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 ter minal fragment of a third isoform of the M110 subunit of phosphatase 1 . This new isoform contains an insert in the 542-597 position not pres ent in the M110 previously cloned (Chen et al, (1994) FEES Lett. 356, 51-55) from the same species, The encoded cDNA was expressed as a solu ble GST-fusion protein in E. coli, and its ability to interact with na tive PP-1C was measured both in vitro and in permeabilized smooth musc le, In vitro, the fusion protein was capable of selectively binding PP -1C and increasing the substrate specificity of the phosphatase toward s myosin 13.2 +/- 3.5-fold (S.E. of the mean, 3). In permeabilized smo oth muscle pretreated with microcystin, the recombinant protein atone (1.0 mu M) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 mu M) 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 specific ity, The presence of isoformic variation within this sequence may perm it organ/cell specific regulation of phosphorylation sites.