Protein Phosphatase-1 Holoenzyme Research Articles

PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309-691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309-691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401-->Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.

GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor.

The cascade of phosphorylation is a pivotal event in transforming growth factor β (TGFβ) signaling. Reversible phosphorylation regulates fundamental aspects of cell activity. TGFβ-induced Smad7 binds to type I receptor (TGFβ type I receptor; TβRI) functioning as a receptor kinase antagonist. We found Smad7 interacts with growth arrest and DNA damage protein, GADD34, a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which subsequently recruits catalytic subunit of PP1 (PP1c) to dephosphorylate TβRI. Blocking Smad7 expression by RNA interference inhibits association of GADD34–PP1c complex with TβRI, indicating Smad7 acts as an adaptor protein in the formation of the PP1 holoenzyme that targets TβRI for dephosphorylation. SARA (Smad anchor for receptor activation) enhances the recruitment PP1c to the Smad7–GADD34 complex by controlling the specific subcellular localization of PP1c. Importantly, GADD34–PP1c recruited by Smad7 inhibits TGFβ-induced cell cycle arrest and mediates TGFβ resistance in responding to UV light irradiation. The dephosphorylation of TβRI mediated by Smad7 is an effective mechanism for governing negative feedback in TGFβ signaling.

Phosphoprotein Inhibitors of Protein Phosphatase-1

The chapter discusses phosphoprotein inhibitors of protein phosphatase-1(PP1). Over 25 years ago, a search for proteins that could regulate phosphorylase phosphatase yielded heat-stable proteins that were called “inhibitor-1” (I-1) and “inhibitor-2” (I-2). I-1 potency as an inhibitor increased several hundred-fold after phosphorylation of Thr35 by cAMP-dependent protein kinase (PKA), whereas inhibitor-2 was effective without prior phosphorylation. Inhibitor-1 became the prototype for other phosphoprotein inhibitors of PP1. To distinguish protein phosphates in type-1 (PP1, sensitive to inhibitors) from type-2, these inhibitor proteins are made use of subsequently.. Several new PP1 inhibitor phosphoproteins, such as NIPP-1, inhibitor-4, CPI-17, PHI-1 and KEPI have been discovered in mammalian cells. Regulation of PP1 in cells incorporates the various inhibitor phosphoproteins to selectively target a subset of PP1 holoenzymes. This way the inhibitor proteins work in concert with the PP1 regulatory subunits. Study of the phosphorylation of the PP1 inhibitors takes on added significance for understanding signaling processes, and the chapter describes various methods developed for that purpose.

Identification of sds21 in fission yeast in an inhibitor-resistant high molecular mass protein phosphatase-1 complex.

While characterizing the type-1 protein phosphatases sds21 and dis2 in fission yeast (Schizosaccharomyces pombe) a novel high molecular mass protein was identified with serine/threonine phosphatase activity (referred to as PP-R) that was resistant to a panel of characteristic inhibitors of protein phosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (deltadis2) resulted predominantly in identification of PP-R. To test the hypothesis that the catalytic activity of PP-R comprised sds21, a parallel purification was performed of PP-1 activity from an S. pombe knockout strain lacking sds21 (deltasds21). Both deltasds21 and deltadis2 strains exhibited similar protein phosphatase activity profiles as determined by DEAE-sepharose, Mono-Q and Superdex gel filtration chromatography. However, the peak of protein phosphatase activity from deltasds21 S. pombe that co-migrated with PP-R from deltadis2 S. pombe exhibited the sensitivity to a panel of inhibitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the resistance to inhibitors may originate from structural differences between dis2 and sds21 isoforms. A key structural feature present in sds21, but lacking in dis2, is a classical phosphorylation consensus sequence surrounding serine-145 of sds21. The previous hypothesis was that PP-1 activity among several lower eukaryotes may be regulated directly by cAMP-dependent protein kinase (PKA) phosphorylation. However, this study demonstrated that recombinant sds21 is not a target for PKA in vitro. The constrained configuration of the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.

Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle.

CPI17, a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory activity is potentiated by protein kinase C and its related enzymes [Eto, M. et al. (1997) FEBS Lett. 410, 356-360]. In order to identify its physiological target in smooth muscle, the myofibrillar extract from porcine aorta media was analyzed by affinity chromatography on CPI17-conjugated Sepharose. The binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated myosin (p-myosin) and phosphorylase-a were bound to the resin and could be eluted with 0.5 M NaCl. The IC50 values of thiophosphorylated CPI17 toward phosphatases bound to the resin were in the range of 0.5-3 nM, as expected for the PP1 holoenzymes sensitive to CPI17. The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chromatography on Mono Q, which suggested multiple functions of CPI17 as a mediator of the protein kinase C-related signal transduction pathway in aorta smooth muscle. The major activity toward p-myosin was identified as the myofibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) was consistent with that of PP1M from chicken gizzard and porcine bladder. The purified PP1M was completely inhibited by phosphorylated and thiophosphorylated CPI17. Kinetic analysis showed mixed inhibition of PP1M by CPI17 (Ki = 1.9 nM and Ki' = 5.1 nM). The concentration of CPI17 in aorta smooth muscle cells was estimated to be at least 0. 3 microM from the result of Western analysis. This concentration appears to be sufficient to suppress the in situ PP1M in aorta smooth muscle, and PP1M is thus identified as a target of CPI17 in vascular smooth muscle.