The control of protein phosphatase‐1 by targetting subunits

Abstract

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130,37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the /3 isoform of protein phosphatase-1 (PPl) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PPlg with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.Although phosphoserine and phosphothreonine account for more than 99% of phosphorylated amino acids [I] and hundreds of phosphoproteins are present in cells, only four major classes of protein serineithreonine phosphatase (PP) catalytic subunits, termed PP1, PP2A, PP2B and PP2C have been identified (reviewed in [2]). Three of these, namely PP1 , PP2A and PP2C, have broad and overlapping substrate specificities in vitro, accounting for most, if not all, of the phosphatase activity in tissue extracts towards a variety of enzymes that control the major metabolic pathways (reviewed in [3]). Furthermore, studies with the tumour promoter okadaic acid, a potent and specific inhibitor of PP1 and PP2A (reviewed in [4]), and the phenotypes of genetic mutants lacking these enzymes in fungi [5], fission yeast [6] and fruit flies [7, 81, has implicated PP1 and PP2A in the regulation of additional processes, such as the transport of ions and nutrients into cells, gene transcription, muscle contractility and the celldivision cycle. The regulation of many physiological processes by a limited number of phosphatase catalytic subunits, has therefore raised the question of how each of these processes can be regulated independently of one another.A major clue originated from studies on thc control of glycogen metabolism in mammalian skeletal muscle, whereCorrespondence to P. Cohen, MRC Protein Phosphorylation Unit,

Amino acid sequence and expression of the hepatic glycogen‐binding (GL‐subunit of protein phosphatase‐1

“…PPlG was purified from rabbit skeletal muscle [26] and the PPl catalytic subunit dissociated from GM by incubation for 2 h in 2 M LiBr and purified by gel filtration on Superose 12 in the presence of 0.5 M LiBr [2]. Sources of other Materials are given in [23].…”

“…For example, the M,,,-subunit which targets PPl to the myofibrils of smooth muscle enhances the rate at which PPl dephosphorylates smooth muscle myosin, but not the rate at which it dephosphorylates skeletal muscle myosin. In contrast, the structurally related M-subunit which targets PPl to the myofibrils of striated muscles, enhances the rate at which PPI dephosphorylates skeletal muscle myosin far more than it enhances the rate of dephosphorylation of smooth muscle myosin [2,3].…”

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

“…therein). At least three laboratories have purified to homogeneity the major form of avian and mammalian smooth muscle myosin phosphatase, SMPP-1M [5][6][7]. By SDS-PAGE analysis, SMPP-1M consists of a heterotrimer of subm~its of 130 kDa, 37 kDa and 20 kDa respectively [5,6].…”

“…At least three laboratories have purified to homogeneity the major form of avian and mammalian smooth muscle myosin phosphatase, SMPP-1M [5][6][7]. By SDS-PAGE analysis, SMPP-1M consists of a heterotrimer of subm~its of 130 kDa, 37 kDa and 20 kDa respectively [5,6]. The 3": kDa subunit has been identified by amino acid sequencing as the catalytic subunit of protein phosphatase 1 [5,6] kDa and 20 kDa subunits can be separated from PP-1C using high concentrations of chaotropic agents.…”

“…By SDS-PAGE analysis, SMPP-1M consists of a heterotrimer of subm~its of 130 kDa, 37 kDa and 20 kDa respectively [5,6]. The 3": kDa subunit has been identified by amino acid sequencing as the catalytic subunit of protein phosphatase 1 [5,6] kDa and 20 kDa subunits can be separated from PP-1C using high concentrations of chaotropic agents. Once separated from these subunits, PP-IC dramatically loses specificity for myosin, regaining it when the subunits are remixed [5,6].…”