Phosphatase Treatment Research Articles

The presence of phosphorylation form of D1 protein in its cross-linked aggregates in high light treated spinach leaves in vivo

In the present study, using specific antibody against D1 protein, we detected four aggregates of D1 protein in thylakoid membranes from spinach leaves illuminated at high light (800–2500 μmol photons·m−2·s−1) for 3 h. Their accumulations were dependent on the light intensity to which the leaves had been subjected. Further immunoblot analysis indicated that 70 kD aggregate was a product of D1 protein cross-linked with CP43, 65 and 60 kD aggregate were two cross-linked products between D1 and D2 proteins, and 41 kD aggregate was one cross-linked D1 with α-subunit of cytochrome b559 (Cyt b559). This result provided the evidence for the existence of the aggregation of the D1 protein in vivo. The maximal level of D1/Cyt b559 aggregate occurred at 1000 μmol photons·m−2·s−1 but drastically decreased with further increasing light intensity. Immunoblot analysis with phosphothreonine (Thr (P)) antibody indicated that D1/CP43 and D1/Cyt b559 aggregates contained the phosphorylated protein(s). In vitro dephosphorylation experiment also showed that D1/Cyt b559 and D1/CP43 aggregates lost the immunoreactivity with Thr (P) antibody after the phosphatase treatment of the membranes from high-light-illuminated leaves. Our results demonstrated that strong illumination of spinach leaves induced cross-linked products of D1 protein with its nearby polypeptides of PS, some of which co.n-tained the phosphorylated D1 protein.

Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium

In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.

Detection of phosphorylation patterns in rat cortical neurons by combining phosphatase treatment and DIGE technology

Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology. About 131 spots were recognized as phosphorylated proteins as they showed different migration behaviour after phosphatase treatment. The analysis of 42 selected spots was carried out by LC/MS/MS technology resulting in the identification of two new phosphoproteins.

Expression and purification of the intact cytoplasmic domain of the human ephrin receptor A2 tyrosine kinase in Escherichia coli

The ephrin receptor A2 (EphA2) is an integral membrane protein tyrosine kinase and a member of the Eph family, the largest known family of receptor tyrosine kinases. EphA2 overexpression is sufficient to transform normal epithelial cells into an aggressive, metastatic phenotype. In normal cells, EphA2 negatively regulates cell growth and invasiveness. Here we report expression of the intact cytoplasmic domain (juxtamembrane linker, tyrosine kinase, and sterile α motif domains) of the human EphA2 receptor in an Escherichia coli system. The expressed protein was purified to near homogeneity by use of metal chelation chromatography combined with removal of vector-encoded tags by specific proteolysis. The cytoplasmic domains of EphA2 are expressed as an active kinase, with the expressed protein found to contain phosphorylated tyrosine residues. In addition, protein tyrosine phosphorylation appears only after EphA2 expression is induced and is removable with alkaline phosphatase treatment. The enzyme was purified 5-fold in yields that average 10–30 mg/L of active EphA2 cytoplasmic domains, which will now be used for further biophysical and structural characterization.

Involvement of Bcl-XL deamidation in E1A-mediated cisplatin sensitization of ovarian cancer cells

The adenovirus E1A protein has been shown to be involved in the potentiation of apoptosis induced by chemotherapeutic agents, yet the molecular events of E1A-mediated apoptosis are not very clear. A recent report has suggested that deamidation of the Bcl-X(L) protein inhibits its antiapoptotic ability and leads to apoptosis induced by alkylating agents in Rb-deficient tumor cells. Since Rb is known to interact with E1A, which interrupts Rb's normal function, we examined Bcl-X(L) deamidation and cell death induced by cisplatin in E1A transfectants. We found that the E1A transfectants became sensitive to cisplatin-induced apoptosis compared to the parental cells, SKOV3.ip1. Our data show that cisplatin treatment induced the modification of Bcl-X(L) in the E1A transfectants in dosage and time-dependent manner. Furthermore, phosphatase treatment had no effect on the level of Bcl-X(L) modification, whereas alkaline lysis buffer appeared to induce the same modification of Bcl-X(L). Ectopic expression of the deamidated forms of Bcl- X(L) in SKOV3.ip1 cells revealed that the modification to the Bcl- X(L) protein molecules was deamidation. Expression of the E1A mutant (dl1108) which contains deletion at CR2 domain suppressed Bcl-X(L) deamidation and apoptosis induced by cisplatin. We also found that expression of the nondeamidated Bcl-X(L) protected E1A transfectants from apoptosis. These findings suggest that Bcl-X(L) deamidation contributes to E1A-mediated cisplatin sensitization in SKOV3.ip1 cells.

DMi-2 Chromatin Binding and Remodeling Activities Are Regulated by dCK2 Phosphorylation

A plethora of ATP-dependent chromatin-remodeling enzymes have been identified during the last decade. Many have been shown to play pivotal roles in the organization and expression of eukaryotic genomes. It is clear that their activities need to be tightly regulated to ensure their coordinated action. However, little is known about how ATP-dependent remodelers are regulated at the molecular level. Here, we have investigated the ATP-dependent chromatin remodeling enzyme Mi-2 of Drosophila melanogaster. Radioactive labeling of S2 cells reveals that dMi-2 is a phosphoprotein in vivo. dMi-2 phosphorylation is constitutive, and we identify dCK2 as a major dMi-2 kinase in cell extracts. dCK2 binds to and phosphorylates a dMi-2 N-terminal region. Dephosphorylation of recombinant dMi-2 increases its affinity for the nucleosome substrate, nucleosome-stimulated ATPase, and ATP-dependent nucleosome mobilization activities. Our results reveal a potential mechanism for regulation of the dMi-2 enzyme and point toward CK2 phosphorylation as a common feature of CHD family ATPases.

ERK1/2 is an endogenous negative regulator of the γsecretase activity

As an essential protease in the generation of amyloid beta, gamma-secretase is believed to play an important role in the pathogenesis of Alzheimer's disease. Although a great deal of progress has been made in identifying the components of gamma-secretase complex, the endogenous regulatory mechanism of gamma-secretase is unknown. Here we show that gamma-secretase is endogenously regulated via extracellular signal regulated MAP kinase (ERK) 1/2-dependent mitogen-activated protein kinase (MAPK) pathway. The inhibition of ERK1/2 activity, either by a treatment with a MEK inhibitor or an ERK knockdown transfection, dramatically increased gamma-secretase activity in several different cell types. JNK or p38 kinase inhibitors had little effect, indicating that the effect is specific to ERK1/2-dependent MAPK pathway. Conversely, increased ERK1/2 activity, by adding purified active ERK1/2 or EGF-induced activation of ERK1/2, significantly reduced gamma-secretase activity, demonstrating down-regulation of gamma-secretase activity by ERK1/2. Whereas gamma-secretase expression was not affected by ERK1/2, its activity was enhanced by phosphatase treatment, indicating that ERK1/2 regulates gamma-secretase activity by altering the pattern of phophorylation. Among the components of isolated gamma-secretase complex, only nicastrin was phosphorylated by ERK1/2, and it precipitated with ERK1/2 in a co-immunoprecipitation assay, which suggests binding between ERK1/2 and nicastrin. Our results show that ERK1/2 is an endogenous regulator of gamma-secretase, which raises the possibility that ERK1/2 down-regulates gamma-secretase activity by directly phosphorylating nicastrin.

Divergent kinase signaling mediates agonist-induced phosphorylation of phosphatase inhibitory proteins PHI-1 and CPI-17 in vascular smooth muscle cells

Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation.

The Essential Role of MEKK3 Signaling in Angiotensin II-induced Calcineurin/Nuclear Factor of Activated T-cells Activation

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes, such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. We determined that active MEKK3 was capable of activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the reduction of NFAT activation in response to angiotensin II in cardiac myocytes. Genetic studies showed that MEKK3-deficient mouse embryo fibroblasts failed to activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. We determined that angiotensin II induced MEKK3 phosphorylation. Thus, MEKK3 functions downstream of the AT1 receptor and is essential for calcineurin/NFAT activation. Finally, we determined that MEKK3-mediated activation of calcineurin/NFAT signaling was associated with the phosphorylation of modulatory calcineurin-interacting protein 1 at Ser(108) and Ser(112). Taken together, our studies reveal a previously unrecognized novel essential regulatory role of MEKK3 signaling in calcineurin/NFAT activation.

Immunoblotting PKC-δ: a cautionary note from the bench

Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC-delta phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC-delta MAb (generally viewed as an anti-PKC-delta protein antibody) recognizes PKC-delta in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC-delta immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC-delta immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC-delta MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC-delta expression in resting and agonist-activated samples (or in studies of the relationship between PKC-delta phosphorylation and PKC-delta downregulation) because it artifactually displays PKC-delta phosphorylation as a decline in total PKC-delta protein. Other studies have shown that two anti-PKC-delta-pY(311) antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC-delta-Y(311) phosphorylation on the endogenous PKC-delta enzyme, but the Cell Signaling Technology anti-PKC-delta-pY(311) antibody provides a better measure of Y(311) phosphorylation in overexpressed PKC-delta. Collectively, these studies have identified features of anti-PKC-delta antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC-delta may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general.

Phosphorylation of Aquaporin PvTIP3;1 Defined by Mass Spectrometry and Molecular Modeling

The water channel protein PvTIP3;1 (alpha-TIP) is a member of the Major Intrinsic Protein membrane channel family. The in vitro activity of this aquaporin is dependent on phosphorylation, and the protein is phosphorylated in vivo by a membrane-associated Ca(2+)-dependent kinase. Mutagenesis studies have implicated three serine residues as kinase targets, but only phosphorylation of Ser7 has been observed in vivo. An atomic model of PvTIP3;1 generated by homology modeling suggested that Ser7 is the only residue that would be sterically accessible to kinases. To further explain the phosphorylation of PvTIP3;1, we overexpressed this aquaporin in the methylotrophic yeast Pichia pastoris and purified the hexahistidine-tagged protein by immobilized metal affinity chromatography. Mass spectrometry confirmed that a fraction of recombinant PvTIP3;1 was phosphorylated. Phosphatase and kinase treatments indicated that Ser7 was the only residue that could be phosphorylated. In addition, mass spectrometry indicated that the native and expressed proteins are N-terminally acetylated. This is the first demonstration that a full-length, recombinant aquaporin can be produced in yeast and authentically phosphorylated in vitro. Characterization of phosphorylation-mediated gating in PvTIP3;1 will serve as a paradigm for understanding gating mechanisms of other channels.

Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

The epithelial cell transforming gene 2 (ECT2) protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. Here we show that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. We identified an in vitro Cdk1 phosphorylation site (T412) in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding was inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, did not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displayed a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant caused cortical hyperactivity in U2OS cells during cell division, this activity was not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert.

Modification of the Stat1 SH2 Domain Broadly Improves Interferon Efficacy in Proportion to p300/CREB-binding Protein Coactivator Recruitment

A normal level of interferon (IFN) responsiveness via the Stat1 transcription factor is critical to the host, since decreased Stat1 signaling causes immune compromise and increased signaling is associated with inflammatory and neoplastic disease. Here we report how this balance may be influenced by novel alterations in the efficiency of Stat1 signaling. To enable disulfide-dependent and spontaneous formation of active Stat1 homodimer (as was done previously for Stat3), we engineered Stat1-CC with double-cysteine substitutions in the Src homology 2 (SH2)-homodimerization domain (at Ala-656 and Asn-658). In this case, however, mutant and wild-type Stat1 exhibited no difference inspontaneousdimerization. Moreover, Stat1-CC still required ligand-dependent Tyr-701 phosphorylation for function and exhibited hyperresponsiveness to IFN-beta (that depends on Stat1/Stat2 heterodimerization) as well as IFN-gamma (that depends on Stat1/Stat1 homodimerization). Hyperresponsivenss of Stat1-CC was accompanied by increased capacities for Tyr-701 phosphorylation and DNA binding, but these features were also found in a similarly substituted serine mutant (Stat1-SS) that showed no hyperresponsiveness to IFN-gamma. This finding raised the possibility that SH2 domain mutations also influence downstream transcriptional efficiency. Indeed, each of these mutations also enhanced recruitment of the normally rate-limiting p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex in proportion to the level of IFN-driven transactivation and gene expression. Additional modifications indicated that the mutant residues in the SH2 domain appeared to cooperate with Ser-727 in the C-terminal domain to regulate p300/CBP interaction with Stat1. The profile of IFN responsiveness translated into the same progressive increase in the level of viral clearance from Stat1- to Stat1-SS- to Stat1-CC-expressing cells. Thus, SH2 domain determinants may be modified to direct better Stat1 phosphorylation, DNA binding, and coactivator recruitment to fully improve IFN efficacy.

Phosphorylation and down-regulation of Bim by muscarinic cholinergic receptor activation via protein kinase C

Bim is one of the proapoptotic BH3-only homologs of the Bcl-2 family proteins, which interacts with other Bcl-2 family proteins to activate the intrinsic apoptotic pathway. The expression and protein level of Bim are highly regulated in cells at both transcriptional and post-translational levels, and inadequate control of Bim level may largely determine its proapoptic activity. In the present study, we reported that carbachol, a muscarinic cholinergic receptor agonist, regulated Bim in human SH-SY5Y neuroblastoma cells. Carbachol rapidly induced an upward gel mobility shift of Bim, which was abolished by protein phosphatase treatment, indicating an increased Bim phosphorylation by carbachol. The effect of carbachol was mimicked by the protein kinase C activator 12-myristate 13-acetate (PMA) and was blocked by the protein kinase C inhibitor rottlerin, suggesting that activation of protein kinase C was required for carbachol-induced phosphorylation of Bim. Prolonged treatment with carbachol and PMA significantly decreased Bim protein levels in total cell lysates and mitrochondria. Carbachol and PMA had no effect in the transcriptional regulation of Bim, whereas the reduction of Bim by both carbachol and PMA was reversed by the proteosome inhibitors, suggesting that carbachol and PMA facilitated the proteosome-dependent Bim degradation. Thus, this study identified the muscarinic receptor-protein kinase C signaling pathway as a regulator of Bim in neuroblastoma cells, and activation of muscarinic receptor and protein kinase C functions to induce Bim phosphorylation, followed by down-regulation of the proapoptotic protein.

Regulation of Yeast mRNA 3′ End Processing by Phosphorylation

Recent studies have found that the phosphatase Glc7 associates with the yeast cleavage/polyadenylation factor (CPF), but the role of Glc7 in 3' end processing has not been investigated. Here, we report that depletion of Glc7 causes shortened poly(A) tails in vivo and accumulation of phosphorylated Pta1, a CPF subunit. Removal of Glc7 also gives extract defective for poly(A) addition but normal for cleavage at the poly(A) site. Polyadenylation is rescued by addition of Glc7 or Pta1, but not by phosphorylated Pta1. Moreover, Ypi1, a Glc7-specific inhibitor, or the Cka1 kinase blocks poly(A) addition in wild-type (wt) extract. Pta1 interacts physically and genetically with Glc7, suggesting that Pta1 may also regulate Glc7 or recruit it to CPF. A weakened association of Fip1 with phosphorylated CPF may explain the specific effect on polyadenylation. These results support a model in which poly(A) synthesis is controlled by cycles of phosphorylation and dephosphorylation that require the action of Glc7.

Ser-557-phosphorylated mCRY2 Is Degraded upon Synergistic Phosphorylation by Glycogen Synthase Kinase-3β

Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.

Cyclic GMP-dependent Protein Kinase Regulates CCAAT Enhancer-binding Protein β Functions through Inhibition of Glycogen Synthase Kinase-3

The CCAAT enhancer-binding protein (C/EBPbeta) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPbeta participates in cGMP-regulated transcription of c-fos in osteoblasts (Chen, Y., Zhuang, S., Cassenaer, S., Casteel, D. E., Gudi, T., Boss, G. R., and Pilz, R. B. (2003) Mol. Cell. Biol. 23, 4066-4082). In the present work, we show that cGMP/cGMP-dependent protein kinase (PKG) induced dephosphorylation and activation of C/EBPbeta by inhibiting glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta on Ser9 negatively regulates the enzyme activity, and we found that PKG phosphorylated this site both in vitro and in vivo; the in vivo phosphorylation occurred rapidly and preceded C/EBPbeta dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3beta is a C/EBPbeta kinase in resting cells. We determined that GSK-3beta phosphorylated C/EBPbeta in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPbeta was phosphorylated on these same sites in intact, unstimulated osteoblasts, and phosphorylation was decreased in cGMP-treated cells. Mutation of the GSK-3 phosphorylation sites in C/EBPbeta prevented C/EBPbeta phosphorylation in resting cells, enhanced C/EBPbeta DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPbeta. cGMP regulation of C/EBPbeta was disrupted by a mutant GSK-3beta(Ala9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPbeta by preventing the negative effects of GSK-3 phosphorylation.

Arrestins in Fungi

Herranz et al . report the identification of a fungal arrestin homolog and its role in pH signaling. PalF, a protein containing C-terminal and N-terminal arrestin domains, interacted with PalH, the 7 transmembrane receptor (7TMR) in two-hybrid assays and glutathione- S -transferase (GST) pull-down assays. The N-terminal arrestin domain was responsible for the interaction with the PalH cytoplasmic domain. Growth at alkaline pH was blocked by truncation of the PalH cytoplasmic domain, which disrupted the interaction with PalF. Shifting of mycelia to alkaline pH resulted in decreased mobility of the PalF protein on SDS-PAGE, which was reversed by phosphatase treatment, consistent with pH-dependent phosphorylation of PalF. Ubiquinated forms of PalF were also detected following the shift to alkaline pH. Both phosphorylation and ubiquitination of PalF were abolished in cells deficient for PalH. Thus, as with metazoan arrestin, fungal arrestin appears to contribute to 7TMR signaling, and this signaling involves similar types of posttranslational modifications as those observed for metazoan arrestin. S. Herranz, J. M. Rodríguez, H.-J. Bussink, J. C. Sánchez-Ferrero, H. N. Arst, Jr., M. A. Peñalva, O. Vincent, Arrestin-related proteins mediate pH signaling in fungi. Proc. Natl. Acad. Sci. U.S.A. 309 , 12141-12146 (2005). [Abstract] [Full Text]

Regulation of Co-repressive Activity of and HDAC Recruitment to RIP140 by Site-specific Phosphorylation

Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs). The N-terminal RD acts by recruiting histone deacetylases (HDACs). In a comprehensive proteomic analysis of RIP140 by MS, 11 phosphorylation sites of RIP140 are identified; among them five sites are located in the N-terminal RD including Ser104, Thr202, Thr207, Ser358, and Ser380. The role of phosphorylation of RIP140 in regulating its biological activity and the underlying mechanism are examined using a site-directed mutagenesis approach. Mutations mimicking constitutive phosphorylation or dephosphorylation are introduced. The N-terminal RD phosphorylation, mediated by the mitogen-activated protein kinase (MAPK), enhances its repressive activity through increased recruitment of HDAC. Mutations mimicking constitutive dephosphorylation at Thr202 or Thr207 significantly impair its repressive activity and HDAC recruitment, whereas mutation at Ser358 only slightly affects its HDAC recruitment and the repressive activity. Consistently, mutations mimicking constitutive phosphorylation at either Thr202 or Thr207 convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr202 and Thr207 residues renders RIP140 fully repressive and strongly interacting with HDAC. The activity of this mutant is resistant to the MAPK inhibitor, indicating an essential role for Thr202 and Thr207 in MAPK-mediated modulation of RIP140 function. The study provides insights into the modulation of RIP140 biological activity through a specific cellular signaling pathway that augments phosphorylation at specific residues of RIP140 molecule and alters its cofactor recruitment.

Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.