Identification of the regions on the M₁₁₀ subunit of protein phosphatase 1M that interact with the M₂₁ subunit and with myosin

We have previously isolated a form of protein phosphatase-l (PP1M) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PP1C) bound to an M-complex consisting of 110-kDa (M₁₁₀) and 21-kDa (M₂₁) subunits. The interaction of PP1C with an N-terminal region of the M₁₁₀ subunit enhances the dephosphorylation of myosin and suppresses the dephosphorylation of other substrates [Alessi, D. R., MacDougall, L. K., Sola, M. M., Ikebe, M. and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035; Chen, Y. H., Chen, M. X., Alessi, D. R., Campbell, D. G., Shanahan, C., Cohen, P. and Cohen, P.T. W. (1994) FEBS Lett. 356, 51-56; Johnson, D. F., Moorhead, G., Caudwell, F. B., Cohen, P., Chen, Y. H., Chen, M. X. and Cohen, P.T. W. (1996) Eur. J. Biochem. 239, 317-325]. In this paper, we establish that PP1M accounts for nearly all the myosin phosphatase activity in myofibrils, that the M₁₁₀ and M₂₁ subunits are present at similar concentrations in the myofibrillar fraction, and that these subunits are entirely bound to PP1. We demonstrate that the M₂₁ subunit does not interact with PP1C, but with the C-terminaI 72 residues of the M₁₁₀ subunit, a region which is 43 % identical to residues 87-161 of the M₂₁ subunit. A fragment of the M₂₁ subunit, M₂₁-(M1-L146), which lacks the C-terminal leucine zipper, also bound to the M₁₁₀ subunit, but two other fragments M₂₁-(M1-E110) and M₂₁-(E110-K186) did not. The M₁₁₀ and M₂₁ subunits were both found to be myosin-binding proteins. The C-terminal 291 residues of the M₁₁₀ subunit, but not the C-terminal 72 residues, bound to myosin, but the N-terminal fragments M₁₁₀-(M1-E309) and M₁₁₀-(M1-S477) did not. Thus, the region of the M₁₁₀ subunit that stimulates the dephosphorylation of myosin by PP1C is distinct from the region that targets PP1M to myosin. Remarkably, each myosin dimer was capable of binding about 20 mol M₂₁ subunit and many of the M₂₁-binding sites were located in the myosin rod domain. The potential significance of this observation is discussed.