The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G‐subunit (GM) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 Å resolution, of PP1c in complex with a 13 residue peptide (GM[63–75]) of GM. The residues in GM[63–75] that interact with PP1c are those in the Arg/Lys–Val/Ile–Xaa–Phe motif that is present in almost every other identified mammalian PP1‐binding subunit. Disrupting this motif in the GM[63–75] peptide and the M110[1–38] peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1‐binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1‐binding proteins whose roles are unknown.
The specificity of the catalytic subunit of protein phosphatase-1 (PP1,) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1,-binding domains on G, and G,, the subunits that target PPI, to hepatic and muscle glycogen, respectively, and on MI,", the subunit that targets PPI, to smooth muscle myosin. G,-(G63 -T93) interacted with PP1, and prevented G, from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate G, from PPI, or affect other characteristic properties of the PPlG, complex. These results indicate that G, contains two PP1,-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, G,-(G63 -N7.5) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates G, from PP1 because phosphate is inserted into the PP1,-binding domain of G,. M,,<)-(Ml -E309) and Ml,,)-(MI -F38), but not Ml,,,-(D39-E309), mimicked the M,,, subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M,,,, subunit and Ml,~l-(MI -E309), neither Ml,,,-(MI -F38) nor M1,,-(D39-E309) suppressed the PPI,-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M,,, subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M,,,-(MI -F38) displaced G, from PPI,, while GM-(G63-T93) displaced M,,,, from PPlC in vitro. These observations indicate that the region(s) of PP1, that interact with G,/G, and MI,, overlap, explaining why different forms of PPI, contain just a single targetting subunit.Keywords: protein phosphatase-1 ; myosin ; smooth muscle ; glycogen metabolism.Protein phosphatase-1 (PPl), one of the major protein serine/ threonine phosphatases of eukaryotic cells, participates in the control of a variety of cellular functions that include glycogen metabolism, muscle contraction, the exit from mitosis (reviewed in [I, 21) and the splicing of mRNA 131. However, evidence has been accumulating that different processes are regulated by ~ Correspondence to P. Cohen, MRC Protein Phosphorylation Unit,
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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