Selective SUMO modification of cAMP-specific phosphodiesterase-4D5 (PDE4D5) regulates the functional consequences of phosphorylation by PKA and ERK

Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, ΨKXE (where Ψ represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of β-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the β2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.

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Targeting of p38 Mitogen-Activated Protein Kinases to MEF2 Transcription Factors

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4028 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4028-4038 ◽

Cited By ~ 172

Author(s):

Shen-Hsi Yang ◽

Alex Galanis ◽

Andrew D. Sharrocks

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Cellular Response ◽

Map Kinases ◽

Biological Response ◽

Extracellular Signals ◽

Extracellular Signal ◽

Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38α and p38β2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).

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Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis

Antioxidants ◽

10.3390/antiox10111833 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1833

Author(s):

Sanja Blaskovic ◽

Yves Donati ◽

Isabelle Ruchonnet-Metrailler ◽

Tamara Seredenina ◽

Karl-Heinz Krause ◽

...

Keyword(s):

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Human Fibroblasts ◽

Cell Types ◽

Post Translational Modification ◽

Pulmonary Tissue ◽

Nadph Oxidase 4

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

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Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.25.2.854-864.2005 ◽

2005 ◽

Vol 25 (2) ◽

pp. 854-864 ◽

Cited By ~ 90

Author(s):

Sandrine Marchetti ◽

Clotilde Gimond ◽

Jean-Claude Chambard ◽

Thomas Touboul ◽

Danièle Roux ◽

...

Keyword(s):

Map Kinases ◽

Fluorescent Protein ◽

Proteasomal Degradation ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Double Mutation ◽

Extracellular Signal ◽

Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

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Fibronectin maintains survival of mouse natural killer (NK) cells via CD11b/Src/β-catenin pathway

Blood ◽

10.1182/blood-2009-05-219881 ◽

2009 ◽

Vol 114 (19) ◽

pp. 4081-4088 ◽

Cited By ~ 22

Author(s):

Ting Zhang ◽

Shuxun Liu ◽

Pengyuan Yang ◽

Chaofeng Han ◽

Jianli Wang ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nuclear Translocation ◽

Nk Cell ◽

Ex Vivo ◽

Interleukin 12 ◽

Erk Phosphorylation ◽

Extracellular Signal ◽

B Cell Leukemia

Abstract Tissue microenvironment and stroma-derived extracellular matrix (ECM) molecules play important roles in the survival and differentiation of cells. Mouse natural killer (NK) cells usually die within 24 hours once isolated ex vivo. Exogenous cytokines such as interleukin-12 (IL-12) and IL-15 are required to maintain the survival and activity of mouse NK cells cultured in vitro. Whether and how ECM molecules such as fibronectin can support the survival of NK cells remain unknown. We demonstrate that fibronectin, just like IL-15, can maintain survival of mouse NK cells in vitro. Furthermore, we show that fibronectin binds to the CD11b on NK cells, and then CD11b recruits and activates Src. Src can directly interact with β-catenin and trigger nuclear translocation of β-catenin. The activation of β-catenin promotes extracellular signal-related kinase (ERK) phosphorylation, resulting in the increased expression of antiapoptotic protein B-cell leukemia 2 (Bcl-2), which may contribute to the maintenance of NK-cell survival. Consistently, fibronectin cannot maintain the survival of CD11b− NK cells and β-catenin–deficient NK cells in vitro, and the number of NK cells is dramatically decreased in the β-catenin–deficient mice. Therefore, fibronectin can maintain survival of mouse NK cells by activating ERK and up-regulating Bcl-2 expression via CD11b/Src/β-catenin pathway.

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Sites of phosphorylation in tau and factors affecting their regulation

Biochemical Society Symposium ◽

10.1042/bss0670073 ◽

2001 ◽

Vol 67 ◽

pp. 73-80 ◽

Cited By ~ 60

Author(s):

Brian H. Anderton ◽

Joanna Betts ◽

Walter P. Blackstock ◽

Jean-Pierre Brion ◽

Sara Chapman ◽

...

Keyword(s):

Glycogen Synthase ◽

Principal Component ◽

Paired Helical Filaments ◽

Glycogen Synthase Kinase 3 ◽

P38 Kinase ◽

Factors Affecting ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Tau Mutation

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.

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Overexpression of RASAL1 indicates poor prognosis and promotes invasion of ovarian cancer

Open Life Sciences ◽

10.1515/biol-2019-0015 ◽

2019 ◽

Vol 14 (1) ◽

pp. 133-140

Author(s):

Rui-Xia Chang ◽

Ai-Ling Cui ◽

Lu Dong ◽

Su-Ping Guan ◽

Ling-Yan Jiang ◽

...

Keyword(s):

Mitogen Activated Protein Kinase ◽

Cell Counting ◽

In Vitro Assays ◽

Ovarian Adenocarcinoma ◽

Invasion And Migration ◽

Kaplan Meier ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

And Migration

AbstractRAS protein activator like-1 (RASAL1) exists in numerous human tissues and has been commonly demonstrated to act as a tumor suppressor in several cancers. This study aimed to identify the functional characteristics of RASAL1 in ovarian adenocarcinoma and a potential mechanism of action. We analyzed RASAL1 gene expression in ovarian adenocarcinoma samples and normal samples gained from the GEO and Oncomine databases respectively. Then the relationship between RASAL1 expression and overall survival (OS) was assessed using the Kaplan-Meier method. Furthermore, the biological effect of RASAL1 in ovarian adenocarcinoma cell lines was assessed by Quantitative real time-PCR (qRT-PCR), Cell Counting Kit-8 (CCK-8), western blot, wound healing and transwell assay. The statistical analysis showed patients with higher RASAL1 expression correlated with worse OS. The in vitro assays suggested knockdown of RASAL1 could inhibit cell proliferation, cell invasion and migration of ovarian adenocarcinoma. Moreover, the key proteins in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway were also decreased in ovarian adenocarcinoma cells with RASAL1 silencing. These findings provide promising evidence that RASAL1 may be not only a powerful biomarker but also an effective therapeutic target of ovarian adenocarcinoma.

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The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

The Journal of Cell Biology ◽

10.1083/jcb.126.4.1059 ◽

1994 ◽

Vol 126 (4) ◽

pp. 1059-1068 ◽

Cited By ~ 125

Author(s):

C Borner ◽

I Martinou ◽

C Mattmann ◽

M Irmler ◽

E Schaerer ◽

...

Keyword(s):

Sympathetic Neurons ◽

Point Mutations ◽

Cell Types ◽

In Vitro Transcription ◽

Site Directed Mutagenesis ◽

Full Activity ◽

Membrane Attachment ◽

Hydrophobic Tail ◽

Critical Regions

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

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IQGAP1 Is a Scaffold for Mitogen-Activated Protein Kinase Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.25.18.7940-7952.2005 ◽

2005 ◽

Vol 25 (18) ◽

pp. 7940-7952 ◽

Cited By ~ 140

Author(s):

Monideepa Roy ◽

Zhigang Li ◽

David B. Sacks

Keyword(s):

Growth Factor ◽

Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Kinase Signaling ◽

Cellular Functions ◽

Molecular Scaffold ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Erk Activity

ABSTRACT IQGAP1 modulates many cellular functions such as cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds extracellular signal-regulated kinase (ERK) 2 and regulates growth factor-stimulated ERK activity. Here we show that MEK, the molecule immediately upstream of ERK in the Ras/mitogen-activated protein (MAP) kinase signaling cascade, also interacts directly with IQGAP1. Both MEK1 and MEK2 bound IQGAP1 in vitro and coimmunoprecipitated with IQGAP1. The addition of ERK2 enhanced by fourfold the in vitro interaction of MEK2 with IQGAP1 without altering binding of MEK1. Similarly, ERK1 promoted MEK binding to IQGAP1, but either MEK protein altered the association between IQGAP1 and ERK. Epidermal growth factor (EGF) differentially regulated binding, enhancing MEK1 interaction while reducing MEK2 binding to IQGAP1. In addition, both knockdown and overexpression of IQGAP1 reduced EGF-stimulated activation of MEK and ERK. Analyses with selective IQGAP1 mutant constructs indicated that MEK binding is crucial for IQGAP1 to modulate EGF activation of ERK. Our data strongly suggest that IQGAP1 functions as a molecular scaffold in the Ras/MAP kinase pathway.

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Phosphorylation of the ARE-binding protein DAZAP1 by ERK2 induces its dissociation from DAZ

Biochemical Journal ◽

10.1042/bj20060681 ◽

2006 ◽

Vol 399 (2) ◽

pp. 265-273 ◽

Cited By ~ 23

Author(s):

Simon Morton ◽

Huei-Ting Yang ◽

Ntsane Moleleki ◽

David G. Campbell ◽

Philip Cohen ◽

...

Keyword(s):

Protein Kinase ◽

Rna Binding ◽

Binding Protein ◽

Mitogen Activated Protein Kinase ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Hek 293 ◽

Extracellular Signal ◽

Mitogen Activated Protein

A protein in RAW 264.7 macrophages, which became phosphorylated in response to LPS (lipopolysaccharide), was identified as the RNA-binding protein called DAZAP1 [DAZ (deleted in azoospermia)-associated protein 1]. The phosphorylation of this protein was prevented by specific inhibition of MKK1 [MAPK (mitogen-activated protein kinase) kinase 1], indicating that it was phosphorylated via the classical MAPK cascade. Further experiments showed that DAZAP1 was phosphorylated stoichiometrically in vitro by ERK2 (extracellular-signal-regulated protein kinase 2) at two Thr-Pro sequences (Thr269 and Thr315), and that both sites became phosphorylated in HEK-293 (human embryonic kidney 293) cells in response to PMA or EGF (epidermal growth factor), or RAW 264.7 macrophages in response to LPS. Phosphorylation induced by each stimulus was prevented by two structurally distinct inhibitors of MKK1 (PD184352 and U0126), demonstrating that DAZAP1 is a physiological substrate for ERK1/ERK2. The mutation of Thr269 and Thr315 to aspartate or the phosphorylation of these residues caused DAZAP1 to dissociate from its binding partner DAZ. DAZ interacts with PABP [poly(A)-binding protein] and thereby stimulates the translation of mRNAs containing short poly(A) tails [Collier, Gorgoni, Loveridge, Cooke and Gray (2005) EMBO J. 24, 2656–2666]. In the present study we have shown that DAZ cannot bind simultaneously to DAZAP1 and PABP, and suggest that the phosphorylation-induced dissociation of DAZ and DAZAP1 may allow the former to stimulate translation by interacting with PABP.

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Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.2.f291 ◽

2001 ◽

Vol 280 (2) ◽

pp. F291-F302 ◽

Cited By ~ 174

Author(s):

Olga Kifor ◽

R. John MacLeod ◽

Ruben Diaz ◽

Mei Bai ◽

Toru Yamaguchi ◽

...

Keyword(s):

Protein Kinase ◽

Combined Treatment ◽

Cell Types ◽

Serine Phosphorylation ◽

Mitogen Activated Protein Kinase ◽

Hek293 Cells ◽

Calcium Sensing ◽

Cytosolic Phospholipase ◽

Extracellular Signal ◽

Mitogen Activated Protein

Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium (Cao 2+)-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating Cao 2+ or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high Cao 2+-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both Gq and Gi. High Cao 2+ increased serine phosphorylation of the 85-kDa cytosolic phospholipase A2(cPLA2) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-Cao 2+-induced ERK1/2 activation and reduced cPLA2 phosphorylation in both cell types, documenting MAPK's role in cPLA2 activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through Gq/11-mediated activation of PI-PLC, as well as through Gi- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in cPLA2 activation.

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