Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.     

A protein phosphatase 2A catalytic subunit is a negative regulator of abscisic acid signalling

The key regulatory role of abscisic acid (ABA) in many physiological processes in plants is well established. However, compared with other plant hormones, the molecular mechanisms underlying ABA signalling are poorly characterized. In this work, a specific catalytic subunit of protein phosphatase 2A (PP2Ac-2) has been identified as a component of the signalling pathway that represses responses to ABA. A loss-of-function pp2ac-2 mutant is hypersensitive to ABA. Moreover, pp2ac-2 plants have altered responses in developmental and environmental processes that are mediated by ABA, such as primary and lateral root development, seed germination and responses to drought and high salt and sugar stresses. Conversely, transgenic plants overexpressing PP2Ac-2 are less sensitive to ABA than wild type, a phenotype that is manifested in all the above-mentioned physiological processes. DNA microarray hybridization experiments reveal that PP2Ac-2 is negatively involved in ABA responses through regulation of ABA-dependent gene expression. Moreover, the results obtained indicate that ABA antagonistically regulates PP2Ac-2 expression and PP2Ac-2 activity thus allowing plant sensitivity to the hormone to be reset after induction. Phenotypic, genetic and gene expression data strongly suggest that PP2Ac-2 is a negative regulator of the ABA pathway. Activity of protein phosphatase 2A thus emerges as a key element in the control of ABA signalling.     

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.     

The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.     

Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis.

Protein phosphatases 2C (PP2Cs) exhibit diverse regulatory functions in signalling pathways of animals, yeast and plants. ABI1 is a PP2C of Arabidopsis that exerts negative control on signalling of the phytohormone abscissic acid (ABA). Characterisation of the redox sensitivity of ABI1 revealed a strong enzymatic inactivation by hydrogen peroxide (H2O2) which has recently been implicated as a secondary messenger of ABA signalling. H2O2 reversibly inhibited ABI1 activity in vitro with an IC(50) of approximately 140 microM in the presence of physiological concentrations of glutathione. In addition, ABI1 was highly susceptible to inactivation by phenylarsine oxide (IC(50)=3-4 microM) indicative for the facile oxidation of vicinal cysteine residues. Thus, H2O2 generated during ABA signalling seems to inactivate the negative regulator of the ABA response.     

A negative regulator of mitosis in Aspergillus is a putative membrane-spanning protein.

The temperature-sensitive cell cycle mutation bimE7 of Aspergillus nidulans causes cells to become blocked in mitosis at a restrictive temperature. Previous work has shown that this mitotic block is induced even when cells are arrested in the S or G2 phase. The mitotic block is also observed in cells carrying a null mutation in bimE, obtained by molecular disruption of the gene (Osmani, S.A., Engle, D.B., Doonan, J.H., and Morris, N.R. (1988) Cell 52, 241-251), indicating that a lack of bimE function is responsible for the phenotype. We have cloned the bimE gene by complementation of the mutant phenotype and have isolated and sequenced its corresponding cDNA. The gene product is encoded by a 6.5-7-kilobase mRNA. The deduced amino acid sequence suggests a protein with three transmembrane domains. The sequence contains numerous potential N-glycosylation sites and several putative cAMP-dependent phosphorylation sites. No homologous protein sequences were found in the common data bases. The bimE gene product is a novel component in the regulation of mitosis.     

Mon2 is a negative regulator of the monomeric G protein, Arl1

Using site-directed mutants of ARL1 predicted to alter nucleotide binding, we examined phenotypes associated with the loss of ARL1 , including effects on membrane traffic and K (+) homeostasis. The GTP-restricted allele, ARL[Q72L] , complemented the membrane traffic phenotype (CPY secretion), but not the K (+) homeostasis phenotypes (sensitivity to hygromycin B, steady-state levels of K (+) , and accumulation of (86) Rb (+) ), while the XTP-restricted mutant, ARL1[D130N] , complemented the ion phenotypes, but not the membrane traffic phenotype. A GDP-restricted allele, ARL1[T32N] , did not effectively complement either phenotype. These results are consistent with a model in which Arl1 has three different conformations in vivo. We also explored the relationship between ARL1 and MON2 using the synthetic lethal phenotype exhibited by these two genes and demonstrated that MON2 is a negative regulator of the GTP-restricted allele of ARL1 , ARL1[Q72L] . Finally, we constructed several new alleles predicted to alter binding of Arl1 to the sole GRIP domain containing protein in yeast, Imh1, and found that ARL1[F52G] and ARL1[Y82G] were unable to complement the loss of ARL1 with respect to either the membrane traffic or K (+) homeostasis phenotypes. Our study expands understanding of the roles of Arl1 in vivo.     

The T cell protein tyrosine phosphatase is a negative regulator of janus family kinases 1 and 3

BACKGROUND: The immune response is regulated through a tightly controlled cytokine network. The counteracting balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activity regulates intracellular signaling in the immune system initiated by these extracellular polypeptides. Mice deficient for the T cell protein tyrosine phosphatase (TCPTP) display gross defects in the hematopoietic compartment, indicating a critical role for TCPTP in the regulation of immune homeostasis. To date, the molecular basis underlying this phenotype has not been reported. RESULTS: We have identified two members of the Janus family of tyrosine kinases (JAKs), JAK1 and JAK3, as bona fide substrates of TCPTP. Inherent substrate specificity in the TCPTP-JAK interaction is demonstrated by the inability of other closely related PTP family members to form an in vivo interaction with the JAKs in hematopoietic cells. In keeping with a negative regulatory role for TCPTP in cytokine signaling, expression of TCPTP in T cells abrogated phosphorylation of STAT5 following interleukin (IL)-2 stimulation. TCPTP-deficient lymphocytes treated with IL-2 had increased levels of tyrosine-phosphorylated STAT5, and thymocytes treated with interferon (IFN)-alpha or IFN-gamma had increased tyrosine-phosphorylated STAT1. Hyperphosphorylation of JAK1 and elevated expression of iNOS was observed in IFN-gamma-treated, TCPTP-deficient, bone marrow-derived macrophages. CONCLUSIONS: We have identified JAK1 and JAK3 as physiological substrates of TCPTP. These results indicate a negative regulatory role for TCPTP in cytokine signaling and provide insight into the molecular defect underlying the phenotype of TCPTP-deficient animals.     

Type 1 phosphatase,a negative regulator of cardiac function

Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired beta-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of beta-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure.     

The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.     

Protein phosphatase 2A is a negative regulator of transforming growth factor-beta1-induced TAK1 activation in mesangial cells

TAK1 (transforming growth factor (TGF)-beta-activated kinase 1) is a serine/threonine kinase that is rapidly activated by TGF-beta1 and plays a vital function in its signal transduction. Once TAK1 is activated, efficient down-regulation of TAK1 activity is important to prevent excessive TGF-beta1 responses. The regulatory mechanism of TAK1 inactivation following TGF-beta1 stimulation has not been elucidated. Here we demonstrate that protein phosphatase 2A (PP2A) plays a pivotal role as a negative regulator of TAK1 activation in response to TGF-beta1 in mesangial cells. Treatment with okadaic acid (OA) induces autophosphorylation of Thr-187 in the activation loop of TAK1. In vitro dephosphorylation assay suggests that Thr-187 in TAK1 is a major dephosphorylation target of PP2A. TGF-beta1 stimulation rapidly activates TAK1 in a biphasic manner, indicating that TGF-beta1-induced TAK1 activation is tightly regulated. The association of PP2A(C) with TAK1 is enhanced in response to TGF-beta1 stimulation and closely parallels TGF-beta1-induced TAK1 activity. Attenuation of PP2A activity by OA treatment or targeted knockdown of PP2A(C) with small interfering RNA enhances TGF-beta1-induced phosphorylation of TAK1 at Thr-187 and MKK3 (MAPK kinase 3). Endogenous TAK1 co-precipitates with PP2A(C) but not PP6(C), another OA-sensitive protein phosphatase, and knockdown of PP6(C) by small interfering RNA does not affect TGF-beta1-induced phosphorylation of TAK1 at Thr-187 and MKK3. Moreover, ectopic expression of phosphatase-deficient PP2A(C) enhances TAK1-mediated MKK3 phosphorylation by TGF-beta1 stimulation, whereas the expression of wild-type PP2A(C) suppresses the MKK3 phosphorylation. Taken together, our data indicate that PP2A functions as a negative regulator in TGF-beta1-induced TAK1 activation.     

T-cell protein tyrosine phosphatase (Tcptp) is a negative regulator of colony-stimulating factor 1 signaling and macrophage differentiation

Mice null for the T-cell protein tyrosine phosphatase (Tcptp-/-) die shortly after birth due to complications arising from the development of a systemic inflammatory disease. It was originally reported that Tcptp-/- mice have increased numbers of macrophages in the spleen; however, the mechanism underlying the aberrant growth and differentiation of macrophages in Tcptp-/- mice is not known. We have identified Tcptp as an important regulator of colony-stimulating factor 1 (CSF-1) signaling and mononuclear phagocyte development. The number of CSF-1-dependent CFU is increased in Tcptp-/- bone marrow. Tcptp-/- mice also have increased numbers of granulocyte-macrophage precursors (GMP), and these Tcptp-/- GMP yield more macrophage colonies in response to CSF-1 relative to wild-type cells. Furthermore, we have identified the CSF-1 receptor (CSF-1R) as a physiological target of Tcptp through substrate-trapping experiments and its hyperphosphorylation in Tcptp-/- macrophages. Tcptp-/- macrophages also have increased tyrosine phosphorylation and recruitment of a Grb2/Gab2/Shp2 complex to the CSF-1R and enhanced activation of Erk after CSF-1 stimulation, which are important molecular events in CSF-1-induced differentiation. These data implicate Tcptp as a critical regulator of CSF-1 signaling and mononuclear phagocyte development in hematopoiesis.     

Suppression of scant identifies Endos as a substrate of greatwall kinase and a negative regulator of protein phosphatase 2A in mitosis

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwl(Scant), a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwl(Scant); many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo(11) partially sterile, even in the absence of gwl(Scant). Heterozygosity for an endos mutation suppresses this PP2A/polo(11) sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwl(Scant) dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop.     

Interaction between protein phosphatase 5 and the A subunit of protein phosphatase 2A: evidence for a heterotrimeric form of protein phosphatase 5

Members of the phosphoprotein phosphatase family of serine/threonine phosphatases are thought to exist in different native oligomeric complexes. Protein phosphatase 2A (PP2A) is composed of a catalytic subunit (PP2Ac) that complexes with an A subunit, which in turn also interacts with one of many B subunits that regulate substrate specificity and/or (sub)cellular localization of the enzyme. Another family member, protein phosphatase 5 (PP5), contains a tetratricopeptide repeat domain at its N terminus, which has been suggested to mediate interactions with other proteins. PP5 was not thought to interact with partners homologous to the A or B subunits that exist within PP2A. However, our results indicate that this may not be the case. A yeast two-hybrid screen revealed an interaction between PP5 and the A subunit of PP2A. This interaction was confirmed for endogenous proteins in vivo using immunoprecipitation analysis and for recombinant proteins by in vitro binding experiments. Our results also indicate that the tetratricopeptide repeat domain of PP5 is required and sufficient for this interaction. In addition, immunoprecipitated PP5 contains associated B subunits. Thus, our results suggest that PP5 can exist in a PP2A-like heterotrimeric form containing both A and B subunits.     

The cystic fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.