Phosphatases: providing safe passage through mitotic exit.

The mitosis-to-interphase transition involves dramatic cellular reorganization from a state that supports chromosome segregation to a state that complies with all functions of an interphase cell. This process, termed mitotic exit, depends on the removal of mitotic phosphorylations from a broad range of substrates. Mitotic exit regulation involves inactivation of mitotic kinases and activation of counteracting protein phosphatases. The key mitotic exit phosphatase in budding yeast, Cdc14, is now well understood. By contrast, in animal cells, it is now emerging that mitotic exit relies on distinct regulatory networks, including the protein phosphatases PP1 and PP2A.

Regulation of Synaptic Transmission and Plasticity by Protein Phosphatase 1.

Protein phosphatases, by counteracting protein kinases, regulate the reversible phosphorylation of many substrates involved in synaptic plasticity, a cellular model for learning and memory. A prominent phosphatase regulating synaptic plasticity and neurologic disorders is the serine/threonine protein phosphatase 1 (PP1). PP1 has three isoforms (α, ß, and γ, encoded by three different genes), which are regulated by a vast number of interacting subunits that define their enzymatic substrate specificity. In this review, we discuss evidence showing that PP1 regulates synaptic transmission and plasticity, as well as presenting novel models of PP1 regulation suggested by recent experimental evidence. We also outline the required targeting of PP1 by neurabin and spinophilin to achieve substrate specificity at the synapse to regulate AMPAR and NMDAR function. We then highlight the role of inhibitor-2 in regulating PP1 function in plasticity, including its positive regulation of PP1 function in vivo in memory formation. We also discuss the distinct function of the three PP1 isoforms in synaptic plasticity and brain function, as well as briefly discuss the role of inhibitory phosphorylation of PP1, which has received recent emphasis in the regulation of PP1 activity in neurons.

The doxorubicin-induced cell motility network is under the control of the ceramide-activated protein phosphatase 1 alpha.

We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models.

The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.

Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle.

Preemptive Activation of the Integrated Stress Response Protects Mice From Diet-Induced Obesity and Insulin Resistance by Fibroblast Growth Factor 21 Induction.

Integrated stress response (ISR) is a signaling system in which phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by stress-specific kinases and subsequent activation of activation transcription factor (ATF) 4 help restore cellular homeostasis following exposure to environmental stresses. ISR activation has been observed in metabolic diseases, including hepatic steatosis (HS), steatohepatitis (SH), and insulin resistance (IR), but it remains unclear whether ISR contributes to disease pathogenesis or represents an innate defense mechanism against metabolic stresses. Constitutive repressor of eIF2α phosphorylation (CReP) is a critical regulatory subunit of the eIF2α phosphatase complex. Here, we show that CReP ablation causes constitutive eIF2α phosphorylation in the liver, which leads to activation of the ATF4 transcriptional program including increased fibroblast growth factor 21 (FGF21) production. Liver-specific CReP knockout (CRePLKO ) mice exhibited marked browning of white adipose tissue (WAT) and increased energy expenditure and insulin sensitivity in an FGF21-dependent manner. Furthermore, CRePLKO mice were protected from high-fat diet (HFD)-induced obesity, HS, and IR. Acute CReP ablation in liver of HFD-induced obese mice also reduced adiposity and improved glucose homeostasis. Conclusion: These data suggest that CReP abundance is a critical determinant for eIF2α phosphorylation and ensuing ISR activation in the liver. Constitutive ISR activation in the liver induces FGF21 and confers protection from HFD-induced adiposity, IR, and HS in mice. Augmenting hepatic ISR may represent a therapeutic approach to treat metabolic disorders.

Differential localizations of protein phosphatase 1 isoforms determine their physiological function in the heart.

Protein phosphatase 1 isoforms α, ß, and γ (PP1α, PP1ß, and PP1γ) are highly homologous in the catalytic domains but have distinct subcellular localizations. In this study, we utilized both primary cell culture and knockout mice to investigate the isoform-specific roles of PP1s in the heart. In both neonatal and adult cardiac myocytes, PP1ß was mainly localized in the nucleus, compared to the predominant presence of PP1α and PP1γ in the cytoplasm. Adenovirus-mediated overexpression of PP1α led to decreased phosphorylation of phospholamban, which was not influenced by overexpression of either PP1ß or PP1γ. Interestingly, only cardiac-specific knockout of PP1ß resulted in increased HDAC7 phosphorylation, consistent with the predominant nuclear localization of PP1ß. Functionally, deletion of either PP1 isoform resulted in reduced fractional shortening in aging mice, however only PP1ß deletion resulted in interstitial fibrosis in mice as early as 3 weeks of age. Deletion of neither PP1 isoform had any effect on pathological cardiac hypertrophy induced by 2 weeks of pressure overload stimulation. Together, our data suggest that PP1 isoforms have differential localizations to regulate the phosphorylation of their specific substrates for the physiological function in the heart.

Antagonistic Effects of p53 and HIF1A on microRNA-34a Regulation of PPP1R11 and STAT3 and Hypoxia-induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells.

BACKGROUND & AIMS: In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a selective advantage under conditions of hypoxia, but little is known about the mediators of this effect. METHODS: Isogenic colorectal cancer (CRC) cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called INH3, HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 human colon cancers. We analyzed data on human colorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of epithelial-to-mesenchymal transition (EMT), or MIR34A or was associated with metastasis. We determined the effects of disruption Mir34a, Mir34b, and Mir34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging. RESULTS: The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34A gene in p53-defective CRC cells, whereas expression of MIR34A was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34A was required for hypoxia-induced EMT, invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34A. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34A reduced activation of STAT3 in p53-deficient CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-deficient CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption of Mir34a, Mir34b, and Mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with TP53 mutations and metastasis to the liver. CONCLUSIONS: HIF1A represses, whereas p53 increases, expression of MIR34A in CRC cells. MIR34A reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia.

Protein Phosphatase-1 Inhibitor-2 Is a Novel Memory Suppressor.

Reversible phosphorylation, a fundamental regulatory mechanism required for many biological processes including memory formation, is coordinated by the opposing actions of protein kinases and phosphatases. Type I protein phosphatase (PP1), in particular, has been shown to constrain learning and memory formation. However, how PP1 might be regulated in memory is still not clear. Our previous work has elucidated that PP1 inhibitor-2 (I-2) is an endogenous regulator of PP1 in hippocampal and cortical neurons (Hou et al., 2013). Contrary to expectation, our studies of contextual fear conditioning and novel object recognition in I-2 heterozygous mice suggest that I-2 is a memory suppressor. In addition, lentiviral knock-down of I-2 in the rat dorsal hippocampus facilitated memory for tasks dependent on the hippocampus. Our data indicate that I-2 suppresses memory formation, probably via negatively regulating the phosphorylation of cAMP/calcium response element-binding protein (CREB) at serine 133 and CREB-mediated gene expression in dorsal hippocampus. Surprisingly, the data from both biochemical and behavioral studies suggest that I-2, despite its assumed action as a PP1 inhibitor, is a positive regulator of PP1 function in memory formation. SIGNIFICANCE STATEMENT: We found that inhibitor-2 acts as a memory suppressor through its positive functional influence on type I protein phosphatase (PP1), likely resulting in negative regulation of cAMP/calcium response element-binding protein (CREB) and CREB-activated gene expression. Our studies thus provide an interesting example of a molecule with an in vivo function that is opposite to its in vitro function. PP1 plays critical roles in many essential physiological functions such as cell mitosis and glucose metabolism in addition to its known role in memory formation. PP1 pharmacological inhibitors would thus not be able to serve as good therapeutic reagents because of its many targets. However, identification of PP1 inhibitor-2 as a critical contributor to suppression of memory formation by PP1 may provide a novel therapeutic target for memory-related diseases.

Growth arrest and DNA damage-inducible 34 regulates liver regeneration in hepatic steatosis in mice.

UNLABELLED: The liver has robust regenerative potential in response to damage, but hepatic steatosis (HS) weakens this potential. We found that the enhanced integrated stress response (ISR) mediated by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) impairs regeneration in HS and that growth arrest and DNA damage-inducible 34 (Gadd34)-dependent suppression of ISR plays a crucial role in fatty liver regeneration. Although mice fed a high-fat diet for 2 weeks developed moderate fatty liver with no increase in eIF2α phosphorylation before 70% hepatectomy, they showed impaired liver regeneration as a result of reduced proliferation and increased death of hepatocytes with increased phosphorylation of eIF2α and ISR. An increased ISR through Gadd34 knockdown induced C/EBP homologous protein (CHOP)-dependent apoptosis and receptor-interacting protein kinase 3-dependent necrosis, resulting in increased hepatocyte death during fatty liver regeneration. Furthermore, Gadd34 knockdown and increased phosphorylation of eIF2α decreased cyclin D1 protein and reduced hepatocyte proliferation. In contrast, enhancement of Gadd34 suppressed phosphorylation of eIF2α and reduced CHOP expression and hepatocyte apoptosis without affecting hepatocyte proliferation, clearly improving fatty liver regeneration. In more severe fatty liver of leptin receptor-deficient db/db mice, forced expression of hepatic Gadd34 also promoted hepatic regeneration after hepatectomy. CONCLUSION: Gadd34-mediated regulation of ISR acts as a physiological defense mechanism against impaired liver regeneration resulting from steatosis and is thus a possible therapeutic target for impaired regeneration in HS.

Characterisation of several ankyrin repeat protein variant 2, a phosphoprotein phosphatase 1-interacting protein, in testis and spermatozoa.

Phosphoprotein phosphatase 1 (PPP1) catalytic subunit gamma 2 (PPP1CC2), a PPP1 isoform, is largely restricted to testicular germ cells and spermatozoa. The key to understanding PPP1 regulation in male germ cells lies in the identification and characterisation of its interacting partners. This study was undertaken to determine the expression patterns of the several ankyrin repeat protein variant 2 (SARP2), a PPP1-interacting protein, in testis and spermatozoa. SARP2 was found to be highly expressed in testis and spermatozoa, and its interaction with human spermatozoa endogenous PPP1CC2 was confirmed by immunoprecipitation. Expression analysis by RT-qPCR revealed that SARP2 and PPP1CC2 mRNA levels were significantly higher in the spermatocyte fraction. However, microscopy revealed that SARP2 protein was only present in the nucleus of elongating and mature spermatids and in spermatozoa. In spermatozoa, SARP2 was prominently expressed in the connecting piece and flagellum, as well as, to a lesser extent, in the acrosome. A yeast two-hybrid approach was used to detect SARP2-interacting proteins and a relevant interaction with a novel sperm-associated antigen 9 (SPAG9) variant, a testis and spermatozoa-specific c-Jun N-terminal kinase-binding protein, was validated in human spermatozoa. Given the expression pattern of SARP2 and its association with PPP1CC2 and SPAG9, it may play a role in spermiogenesis and sperm function, namely in sperm motility and the acrosome reaction.

PP1γ functionally augments the alternative splicing of CaMKIIδ through interaction with ASF.

Protein phosphatase 1 (PP1) and Ca2+/calmodulin-dependent protein kinase δ (CaMKIIδ) are upregulated in heart disorders. Alternative splicing factor (ASF), a major splice factor for CaMKIIδ splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKIIδ in cells. We found that 1) PP1γ, but not α or ß isoform, enhanced the splicing of CaMKIIδ in HEK293T cells; 2) PP1γ promoted the function of ASF, evidenced by the existence of ASF-PP1γ association as well as the PP1γ overexpression- or silencing-mediated change in CaMKIIδ splicing in ASF-transfected HEK293T cells; 3) CaMKIIδ splicing was promoted by overexpression of PP1γ and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; 4) CaMKIIδ splicing and enhancement of ASF-PP1γ association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1γ and suppressed by inhibition of PP1γ with I1PP1 or tautomycetin in primary cardiomyocytes; 5) functionally, overexpression and inhibition of PP1γ, respectively, potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; and 6) CaMKII was required for the OGD/R induced- and PP1γ exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1γ-treated cells. In conclusion, our results show that PP1γ promotes the alternative splicing of CaMKIIδ through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.

Regulation of metabotropic glutamate receptor 7 (mGluR7) internalization and surface expression by Ser/Thr protein phosphatase 1.

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Phosphorylation of the transcription factor Sp4 is reduced by NMDA receptor signaling.

The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites.

Dopamine D4 receptor transmission in the prefrontal cortex controls the salience of emotional memory via modulation of calcium calmodulin-dependent kinase II.

Dopamine (DA) signaling in the medial prefrontal cortex (mPFC) plays a critical role in the processing of emotional information and memory encoding. Activation of DA D4 receptors within the prelimbic (PLC) division of the mPFC bidirectionally modulates emotional memory by strongly potentiating the salience of normally nonsalient emotional memories but blocking the acquisition of suprathreshold emotionally salient fear memories. Previous in vitro studies have shown that activation of cortical DA D4 receptors can bidirectionally modulate levels of α-calcium calmodulin-dependent kinase II (α-CaMKII), a molecule essential for learning and memory. Using an olfactory fear conditioning procedure in rats combined with microinfusions into the mPFC, we examined the potential role of D4 receptor-mediated control of emotional memory salience through signaling via CaMKII, cAMP/protein kinase A (PKA), and protein phosphatase-1 (PP1) signaling. We report that CaMKII blockade prevents the ability of intra-mPFC DA D4 receptor activation to potentiate the salience of subthreshold fear memory. In contrast, blockade of either cAMP/PKA or PP1 signaling pathways rescued the blockade of suprathreshold fear memory via intra-mPFC D4 receptor activation. Our results demonstrate that modulation of emotional memory salience via intra-mPFC DA D4 receptor transmission depends upon downstream signaling via CaMKII, cAMP/PKA, and PP1 substrates.

p38 mitogen-activated protein kinase mediates hyperosmolarity-induced vasoconstriction through myosin light chain phosphorylation and actin polymerization in rat aorta.

Hyperosmotic stress induces the contractile response of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that cytoskeleton reorganization and Rho/Rho-kinase-mediated inactivation of myosin light chain phosphatase (MLCP) play an important role in hyperosmotic vasoconstriction, but the precise mechanism is unknown. This study aimed to investigate the contractile response of endothelium-denuded rings of rat aortas to hyperosmolar sucrose (160 mM) in the presence or absence of inhibitors for various protein kinases. We found that the hyperosmotic constriction of aortic rings was attenuated not only by ML-7 or hydroxyfasudil, specific inhibitor for myosin light chain kinase (MLCK) or Rho-kinase, respectively, but also by SB203580, a specific inhibitor for p38 mitogen-activated kinase (p38 MAPK). Hyperosmolar sucrose evoked a transient increase in cytosolic free Ca(2+) in rat VSMCs, and this response was not affected by SB203580. Western blot analysis of proteins extracted from rings showed that the hyperosmolar sucrose stimulated phosphorylation of the Rho-kinase-mediated myosin phosphatase target subunit 1, myosin light chain (MLC), and p38 MAPK. The experiments performed using a combination of the kinase inhibitors showed that hyperosmolarity-induced MLC phosphorylation is partially mediated via the SB203580-sensitive pathway and is independent of both MLCK and Rho-kinase-mediated inactivation of MLCP. Furthermore, the hyperosmolarity-induced increase in the F-actin/G-actin ratio in rings was attenuated not only by hydroxyfasudil but also by SB203580. These results suggest that p38 MAPK is involved in hyperosmotic vasoconstriction via stimulation of MLC phosphorylation and cytoskeleton reorganization through pathways independent of activation of MLCK and/or Rho-kinase-mediated mechanisms.

Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease.

BACKGROUND: Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. OBJECTIVE: To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. METHODS: Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. RESULTS: A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions--for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. CONCLUSIONS: Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

Psychosine induces the dephosphorylation of neurofilaments by deregulation of PP1 and PP2A phosphatases.

Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process.

Reg1 protein regulates phosphorylation of all three Snf1 isoforms but preferentially associates with the Gal83 isoform.

The phosphorylation status of the Snf1 activation loop threonine is determined by changes in the rate of its dephosphorylation, catalyzed by the yeast PP1 phosphatase Glc7 in complex with the Reg1 protein. Previous studies have shown that Reg1 can associate with both Snf1 and Glc7, suggesting substrate binding as a mechanism for Reg1-mediated targeting of Glc7. In this study, the association of Reg1 with the three Snf1 isoforms was measured by two-hybrid analysis and coimmunoprecipitation. We found that Reg1 association with Snf1 occurred almost exclusively with the Gal83 isoform of the Snf1 complex. Nonetheless, Reg1 plays an important role in determining the phosphorylation status of all three Snf1 isoforms. We found that the rate of dephosphorylation for isoforms of Snf1 did not correlate with the amount of associated Reg1 protein. Functional chimeric ß subunits containing residues from Gal83 and Sip2 were used to map the residues needed to promote Reg1 association with the N-terminal 150 residues of Gal83. The Gal83 isoform of Snf1 is the only isoform capable of nuclear localization. A Gal83-Sip2 chimera containing the first 150 residues of Gal83 was able to associate with the Reg1 protein but did not localize to the nucleus. Therefore, nuclear localization is not required for Reg1 association. Taken together, these data indicate that the ability of Reg1 to promote the dephosphorylation of Snf1 is not directly related to the strength of its association with the Snf1 complex.