Autophosphorylation of Arabidopsis nucleoside diphosphate kinase 2 occurs only on its active histidine residue

Arabidopsis nucleoside diphosphate kinase 2 (NDPK2) is a component in the phytochrome-mediated light signaling. In the present study, its autophosphorylation was investigated. Acid-stable and alkali-stable phosphorylated residues were analyzed under two different conditions. Results revealed that NDPK2 is phosphorylated only on its active histidine residue His197 and the presence of serine/threonine phosphorylation is an experimental artifact due to the harsh condition applied in the treatment of the phosphorylated protein sample. To resolve the controversy of whether serine/threonine phosphorylation of NDPK occurs as has been suggested by other NDPK studies, NDPK2 putative phosphorylation site mutants were generated and examined. No serine/threonine phosphorylation was identified in NDPK2 or implicated in its enzymatic activity. Further studies indicated that the low enzymatic activity and autophosphorylation level of NDPK2 mutant S199A are shown to be due to a damaged H-bonding with the active histidine residue His197 in the nucleotide-binding pocket. In addition, NDPK2 Kpn loop mutant T182A was found to possess an extremely low enzymatic activity and almost no autophosphorylation, suggesting the importance of the oligomeric states of NDPK2 in NDPK2 functioning.     

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.     

Insulin and platelet-derived growth factor stimulate phosphorylation of the c-raf product at serine and threonine residues in intact cells

We have examined the phosphorylation of the serine threonine kinase, the product of c-raf proto-oncogene in response to insulin or platelet-derived growth factor in intact cells. Both insulin and platelet-derived growth factor stimulated phosphorylation of the c-raf protein about 2- to 3-fold. The phosphorylation occurred exclusively on serine and threonine residues; phosphotyrosine was not detected. In immune-complex kinase assays, treatment with insulin, and platelet-derived growth factor increased autophosphorylation of the c-raf kinase, suggesting activation of its kinase activity. To investigate whether the phosphorylation of the c-raf protein in intact cells results from an autophosphorylation event or from the phosphorylation by other cellular kinase(s), we replaced lysine 375 in the putative ATP-binding domain of the c-raf protein with alanine using oligonucleotide site-directed mutagenesis and expressed the mutated protein in NIH3T3 cells. The substitution resulted in the inactivation of the serine/threonine-specific autophosphorylation in immune-complex kinase assays. In intact cells, however, although phosphorylation of the mutant protein in response to insulin and platelet-derived growth factor occurred to a lesser extent than that of the wild-type protein, the phosphopeptide maps were indistinguishable. These results suggest that serine threonine phosphorylation might be responsible for the activation of c-raf kinase upon treatment of cells with insulin and platelet-derived growth factor, and most of the phosphate associated with the c-raf protein results from its phosphorylation by as yet uncharacterized cellular serine/threonine kinase(s).     

Growth arrest induced by transforming growth factor beta 1 is accompanied by protein phosphatase activation in human keratinocytes.

Protein phosphorylation and dephosphorylation are involved in regulation of cell growth. We tested the hypothesis that the growth inhibitory effect of transforming growth factor beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human keratinocytes in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured keratinocytes with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-2, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type 2A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with growth arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of growth arrest in keratinocytes by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a growth-arrested state.     

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.     

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.     

Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.     

Autophosphorylation of Akt at threonine 72 and serine 246. A potential mechanism of regulation of Akt kinase activity

Activation of the serine/threonine protein kinase Akt is a multistep process. We here propose that the kinase activity of Akt is regulated via autophosphorylation in trans at two putative sites (threonine 72 and serine 246) that lie in the characteristic Akt substrate motif (RXRXX(S/T)). Incubation of Akt immunoprecipitated from transfected cells with a pre-activated Akt recombinant protein and gamma-32P-labeled ATP led to marked incorporation of radioactivity in wild-type Akt but not Akt/T72A/S246A mutant. Western blot analysis using a phosphorylated Akt substrate-specific antibody of Akt immunoprecipitated from transfected cells confirmed the autophosphorylation of wild-type Akt but not Akt/T72A/S246A mutant in insulin-like growth factor-1 (IGF-1)-stimulated cells. Autophosphorylation of Akt on Thr-72 and Ser-246 appeared to require prior phosphorylation of Akt on Thr-308 and Ser-473. Compared with wild-type Akt, Akt/T72A/S246A mutant exhibited markedly reduced basal Akt kinase activity and response to cellular stimulation by insulin-like growth factor-1, and also conferred less cellular resistance to doxorubicin-induced apoptosis. The findings from these pilot studies suggest that Akt regulates its kinase activity through autophosphorylation. Further investigation of this potential novel regulatory mechanism by which Akt performs its cellular functions is warranted.     

Identification of the conserved serine/threonine residues important for gibberellin-sensitivity of Arabidopsis RGL2 protein

The DELLA proteins GAI, RGA, RGL1 and RGL2 in Arabidopsis are plant growth repressors, repressing diverse developmental processes. Studies have shown that gibberellin (GA) attenuates the repressive function of DELLA proteins by triggering their degradation via the proteasome pathway. However, it is not known if GA-induced protein degradation is the only pathway for regulating the bioactivity of DELLA proteins. We show here that tobacco BY2 cells represent a suitable system for studying GA signaling. RGL2 exists in a phosphorylated form in BY2 cells. RGL2 undergoes GA-induced degradation, and this process is blocked by proteasome inhibitors and serine/threonine phosphatase inhibitors; however, serine/threonine kinase inhibitors had no detectable effect, suggesting that dephosphorylation of serine/threonine is probably a prerequisite for degradation of RGL2 via the proteasome pathway. Site-directed substitution of all 17 conserved serine and threonine residues showed that six mutants (RGL2(S441D, RGL2(S542D), RGL2(T271E), RGL2(T319E), RGL2(T411E) and RGL2(T535E)) mimicking the status of constitutive phosphorylation are resistant to GA-induced degradation. This suggests that these sites are potential phosphorylation sites. A functional assay based on the expression of GA 20-oxidase revealed that RGL2(T271E) is probably a null mutant, RGL2(S441D), RGL2(S542D), RGL2(T319E) and RGL2(T411E) only retained about 4-17% of the activity of the wild type RGL2, whereas RGL2(T535E) retained about 66% of the activity of the wild type RGL2. However, expression of GA 20-oxidase in BY2 cells expressing these mutant proteins is still responsive to GA, suggesting that the stabilization of RGL2 protein is not the only pathway for regulating its bioactivity.     

Heat shock induced proteins in plant cells

Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells.     

Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modification

The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.     

Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites

The endothelial isoform of nitric oxide synthase (eNOS) is a calcium/calmodulin-dependent enzyme that catalyzes the synthesis of nitric oxide, a key mediator of vascular homeostasis. eNOS undergoes a variety of posttranslational modifications, including phosphorylation on at least three residues: serines 116 and 1179 and threonine 497. Although the agonist-modulated protein kinase pathways that lead to eNOS phosphorylation have been studied in detail, the signaling pathways governing eNOS dephosphorylation remain less well characterized. The present study identifies protein phosphatase 2A (PP2A) as a key determinant of eNOS dephosphorylation and enzyme activity. We transfected bovine aortic endothelial cells (BAEC) with epitope-tagged cDNAs encoding wild-type eNOS or a series of phosphorylation-deficient eNOS mutants, immunoprecipitated [(32)P(i)] biosynthetically labeled recombinant proteins using antibodies directed against the epitope tag and treated the [(32)P(i)]-phosphorylated eNOS with protein phosphatases. We found that PP2A dephosphorylates eNOS residues threonine 497 and serine 1179 but not serine 116 and that an eNOS mutant lacking these three established phosphorylation sites is robustly labeled when expressed in BAEC and is dephosphorylated by PP2A. An inhibitor of PP2A increases eNOS enzymatic activity and augments overall levels of eNOS phosphorylation, specifically increasing phosphorylation of serines 116 and 1179. When transfected into BAEC or COS-7 cells, a "phospho-mimetic" eNOS mutant in which threonine 497 is changed to aspartate shows attenuated phosphorylation at serine 1179 as well as reduced enzyme activity in COS-7 cells. Our results indicate that regulation of eNOS dephosphorylation may be a key point for control of nitric oxide-dependent signaling pathways in vascular endothelial cells.     

Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

CHRK1, a chitinase-related receptor-like kinase in tobacco

A cDNA encoding a chitinase-related receptor-like kinase, designated CHRK1, was isolated from tobacco (Nicotiana tabacum). The C-terminal kinase domain (KD) of CHRK1 contained all of the conserved amino acids of serine/threonine protein kinases. The putative extracellular domain was closely related to the class V chitinase of tobacco and to microbial chitinases. CHRK1 mRNA accumulation was strongly stimulated by infection with fungal pathogen and tobacco mosaic virus. Amino acid-sequence analysis revealed that the chitinase-like domain of CHRK1 lacked the essential glutamic acid residue required for chitinase activity. The recombinant chitinase-like domain did not show any catalytic activity for either oligomeric or polymeric chitin substrates. The recombinant KD of CHRK1 exhibited autophosphorylation, but the mutant KD with a mutation in the essential ATP-binding site did not, suggesting that CHRK1 encoded a functional kinase. CHRK1 was detected in membrane fractions of tobacco BY2 cells. Furthermore, CHRK1-GFP fusion protein was localized in plasma membranes when it was expressed in animal cells. This is the first report of a new type of receptor-like kinase containing a chitinase-like sequence in the putative extracellular domain.     

Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells

In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.     

Increased protein kinase C activity is linked to reduced insulin receptor autophosphorylation in liver of starved rats

Phosphorylation of the insulin receptor beta-subunit on serine/threonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor beta-subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serine/threonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor beta-subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and cAMP-dependent protein kinase A in liver were studied. A 2-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.     

Confluence induced threonine41/serine45 phospho-β-catenin dephosphorylation via ceramide-mediated activation of PP1cγ

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101–11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-β-catenin at threonine41/serine45. The effect of confluence on β-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of β-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented β-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cγ prevented dephosphorylation of β-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cγ to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced β-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.