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The biotin identification (BioID) protocol uses a mutant of the biotin ligase BirA (BirA*) fused to a protein-of-interest to biotinylate proximate proteins in intact cells. Here, we show that two inactive halves of BirA* separately fused to a catalytic and regulatory subunit of protein phosphatase PP1 reconstitute a functional BirA* enzyme upon heterodimerization of the phosphatase subunits. We also demonstrate that this BirA* fragment complementation approach, termed split-BioID, can be used to screen for substrates and other protein interactors of PP1 holoenzymes. Split-BioID is a novel and versatile tool for the identification of (transient) interactors of protein dimers. [ABSTRACT FROM AUTHOR]

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 ( PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B. [ABSTRACT FROM AUTHOR]

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. [ABSTRACT FROM AUTHOR]

Abstract: AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. In pancreatic beta cells, glucose induces the dephosphorylation of Thr172 within the catalytic subunit and the inactivation of the AMPK complex. Here we demonstrate that glucose also activates protein kinase A (PKA), leading to the phosphorylation of AMPKα at Ser485 and Ser497. However, these modifications do not impair the phosphorylation of Thr172 by upstream kinases, and phosphorylation of Thr172 does not affect the phosphorylation of AMPKα by PKA either. Thus, although phosphorylation of Thr172 and Ser485/Ser497 are inversely correlated in response to glucose, they follow an independent regulation. [Copyright &y& Elsevier]

Mammalian AMP‐activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status. It is activated by phosphorylation of the catalytic subunit on Thr172. The main objective of this study was the identification of a phosphatase involved in the regulation of AMPK activity. Mouse MIN6 β cells were used to study the glucose‐induced regulation of the phosphorylation of AMPK. Small interfering RNA (siRNA) technology was used to deplete putative phosphatase candidate genes that could affect AMPK regulation. The effect of the siRNAs used in the study was compared with the effect observed using a negative control siRNA. A protein phosphatase complex composed of the catalytic subunit of protein phosphatase‐1 (PP1) and the regulatory subunit R6 participates in the glucose‐induced dephos‐phorylation of AMPK. R6 interacts physically with the β ‐subunit of the AMPK complex and recruits PP1 to dephosphorylate the catalytic α‐subunit on Thr172. siRNA depletion of R6 decreases glucose‐induced insulin secretion due to the presence of a constitutively active AMPK complex. The characterization of the PP1‐R6 complex identifies this holoenzyme as a possible target for therapeutic intervention with the aim of regulating the activity of AMPK in pancreatic β cells.— Garcia‐Haro, L., Garcia‐Gimeno, M. A., Neumann, D., Beullens, M., Bollen, M., Sanz, P. The PP1‐R6 protein phosphatase holoenzyme is involved in the glucose‐induced dephosphorylation and inactivation of AMP‐activated protein kinase, a key regulator of insulin secretion, in MIN6 β cells. FASEB J. 24, 5080–5091 (2010). www.fasebj.org [ABSTRACT FROM AUTHOR]

Eukaryotic cells sense decreased oxygen levels and respond by altering their metabolic strategy to sustain non-respiratory ATP production through glycolysis, and thus promote cell survival in a hypoxic environment. Protein phosphatase 1 (PP1) has been recently implicated in the governance of the rational use of energy when metabolic substrates are abundant and contributes to cellular recovery following metabolic stress. Under conditions of hypoxia, the expression of the gamma isoform of PP1 (PP1γ), is diminished, an event we have hypothesized to be involved in the adaptive cellular response to hypoxia. Decreased PP1γ activity in hypoxia has a profound impact on the activity of the cAMP response element binding protein (CREB), a major transcriptional regulator of metabolic genes and processes. Here, we demonstrate a further mechanism leading to inhibition of PP1 activity in hypoxia which occurs at least in part through increased association with the nuclear inhibitor of PP1 (NIPP1), an event dependent upon decreased basal cAMP/PKA-dependent signaling. Using a dominant negative NIPP1 construct, we provide evidence that NIPP1 plays a major role in the regulation of both CREB protein expression and CREB-dependent transcription in hypoxia. Furthermore, we demonstrate functional sequellae of such events including altered gene expression and recovery of cellular ATP levels. In summary, we demonstrate that interaction with NIPP1 mediates decreased PP1γ activity in hypoxia, an event which may constitute an inherent part of the cellular oxygen-sensing machinery and may play a role in physiologic adaptation to hypoxia. J. Cell. Physiol. 209: 211–218, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1α, PP1β and PP1γ1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1γ1 and PP1β were predominantly targeted to the nucleoli, while PP1α showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1α, Arg19 for PP1β and Arg20 for PP1γ1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1β and PP1γ1 harbours an interaction site for one or more nucleolar interactors. [Copyright &y& Elsevier]

Examines the structure and splice products of the human gene encoding sds22. Description of sds22; Role of sds22 in the completion of mitosis in yeast; Component of sds22.

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase- I (PP-1) and protein phosphatase-2A (PP-2A). However. after repeated washings only PP-1 remained bound to the ribosomes. The activity of ribosome-associated PP-1 (PP-1R) was partially latent and could be increased 2-3-fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type-I catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP-IR. We have purified from a ribosomal extract a basic polypeptide (pI ⩾ 10.5) of 23 kDa that potently inhibited PP-I. This ribosomal inhibitor of PP-I. termed RIPP-1. was at least 30-times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP-2A. PP-2B and PP-2C). RIPP-1 was identified as a non-competitive inhibitor of PP-I with a substrate-dependent potency. The lowest K, (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type-1 catalytic subunit. RIPP- I also converted the catalytic subunit in a time-dependent manner (t1/2 = 45 mm at 25°C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP- 1. We propose that RIPP- I accounts, at least in part. for the latency of PP-IR. [ABSTRACT FROM AUTHOR]

When glucose is added to cells of the yeast Saccharomyces cerevisiae grown on non-fermentable carbon sources, a cAMP signal is induced which triggers a protein phosphorylation cascade. Addition of glucose or fructose to cells of a phosphoglucose isomerase mutant also induced the cAMP signal indicating that metabolization of the sugar beyond the sugar phosphate step is not necessary. Glucose 6-phosphate might stimulate the triggering reaction since induction with fructose shows a significant delay. Experiments with double and triple mutants in hexokinase 1, hexokinase 2 or glucokinase indicated that the presence of one of the three kinases was both necessary and enough for induction of the cAMP signal by glucose and the presence of one of the two hexokinases necessary and enough for induction by fructose. The product of the kinase reaction itself however does not appear to be the trigger of the reaction: when the increase in the level of glucose 6-phosphate and fructose 6-phosphate was measured as a function of time after addition of different glucose concentrations, no correlation was observed with the increase in the cAMP level. From the dependence of the cAMP increase on the external concentration of glucose, a rough estimate was obtained of the Km of the triggering reaction: about 25 mM. This value clearly fits with the Km of the low-affinity glucose carrier (about 20 mM) and differs by at least an order of magnitude from the Km values of the high-affinity glucose carrier and the three kinases. The present results situate the primary triggering reaction at the level of transport-associated phosphorylation. The main (= low-affinity) glucose carrier appears to be the receptor while association of the corresponding kinase is needed for induction of the signal. Since it is known that the presence of the kinases influences the characteristics of sugar transport, no definite conclusion can be given on whether the necessity of the kinases reflects the need for a certain type of transport or the need for phosphorylation of the sugar. The increase in the level of fructose 1,6-bisphosphate, on the other hand, correlated very well with the cAMP increase. However, it clearly lagged behind the cAMP increase, confirming the previously suggested importance of the cAMP signal for the stimulation of glycolytic flux at the level of phosphofructokinase 1. The importance of the cAMP signal for the stimulation of phosphofructokinase 1 also provides an explanation for the transient overshoot in the levels of glucose 6-phosphate and fructose 6phosphate which are observed after addition of glucose to derepressed yeast cells. Addition of glucose to glucoserepressed wild-type cells triggers no or just a very weak cAMP signal, indicating that one of the intermediates in the induction sequence must be glucose repressible. This conclusion was confirmed by experiments with wild-type cells grown on either galactose or maltose (which have less glucose repression) and with cells of the hxk2 mutant which is deficient in glucose repression. [ABSTRACT FROM AUTHOR]