Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

Smolenski, Albert; Bachmann, Christiane; Reinhard, Kathrin; Hönig-Liedl, Petra; Jarchau, Thomas; Hoschuetzky, Heinz; Walter, Ulrich

doi:10.1074/jbc.273.32.20029

Cited by 312 publications

(332 citation statements)

References 31 publications

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“…To control the specificity of the Epac-specific activator 8-pCPT-2 0 -O-Me-cAMP (8-pCPT), we analysed the phosphorylation of vasodilatoractivated phosphoprotein (VASP), which is known to be phosphorylated by PKA (Smolenski et al, 1998). Activation of the adenylyl cyclase by forskolin (100 mM) and treatment with the cAMP analogue dbcAMP (1 mM) induced VASP phosphorylation in both cell lines through PKA.…”

Section: Resultsmentioning

confidence: 99%

Epac inhibits migration and proliferation of human prostate carcinoma cells

Grandoch¹,

Rose

Braak³

et al. 2009

Br J Cancer

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BACKGROUND: It was recently found that cAMP mediates protein kinase A-independent effects through Epac proteins. The aim of this study was to investigate the role of Epac in migration and proliferation of prostate carcinoma cells. METHODS: The effect of Epac activation was determined by [ 3 H]thymidine incorporation and scratch assays in PC-3 and DU 145 cells. Furthermore, cytoskeletal integrity was analysed by phalloidin staining. The participation of intracellular Epac effectors such as mitogen-activated protein (MAP) kinases, Rap1-and Rho-GTPases was determined by immunoblotting and pull-down assay. RESULTS: The specific Epac activator 8-pCPT-2 0 -O-Me-cAMP (8-pCPT) interfered with cytoskeletal integrity, reduced DNA synthesis, and migration. Although 8-pCPT activated Rap1, it inhibited MAP kinase signalling and RhoA activation. These findings were translated into functional effects such as inhibition of mitogenesis, cytoskeletal integrity, and migration. CONCLUSION: In human prostate carcinoma cells, Epac inhibits proliferative and migratory responses likely because of inhibition of MAP kinase and RhoA signalling pathways. Therefore, Epac might represent an attractive therapeutic target in the treatment of prostate cancer.

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Section: Resultsmentioning

confidence: 99%

Epac inhibits migration and proliferation of human prostate carcinoma cells

Grandoch¹,

Rose

Braak³

et al. 2009

Br J Cancer

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“…The initial phosphorylation at serine 239 (hereafter called VASP-1P) depends on cGMP-dependent protein kinase, whereupon a second phosphorylation at serine 157 (VASP-2P) can occur through cGMP-or cAMP-dependent kinases, and this causes a shift in the mobility of the protein to a higher apparent molecular weight in SDS-PAGE gels (28). In response to 50 nM NO, VASP-1P was detected at the earliest time points (2-and 5-s exposure), coincident with the rise in cGMP (Fig.…”

Section: Expression Of Gc-coupled No Receptor Subunit Mrna Inmentioning

confidence: 99%

Kinetics of a Cellular Nitric Oxide/cGMP/Phosphodiesterase-5 Pathway

Amin

Bianco³

et al. 2004

Journal of Biological Chemistry

103

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Rat platelets served as a model to evaluate quantitatively how guanylate cyclase (GC)-coupled nitric oxide (NO) receptors and phosphodiesterases (here phosphodiesterase-5) interact to transduce NO signals in cells. The platelets expressed mRNA only for the ␣ 1 and ␤ 1 GC-coupled receptor subunits. In intact platelets, the potency of NO for elevating cGMP (EC 50 ‫؍‬ 10 nM) was lower than in lysed platelets (EC 50 ‫؍‬ 1.7 nM). The limiting activities of GC and phosphodiesterase in intact platelets were both very high, being equivalent to about 100 M/s. With low phosphodiesterase activity (imposed by 100 M sildenafil), the cGMP response over time was hyperbolic in shape for a range of NO concentrations or GC activities due to GC desensitization. Without a phosphodiesterase inhibitor, NO generated only brief cGMP transients, peaking after 2-5 s but amounting maximally to about 150 M cGMP. The transients were caused partly by GC desensitization, which varied in rate (halftime up to 3 s) and extent (up to 80%) depending on the NO concentration, and partly by an enhancement of the phosphodiesterase catalytic activity with time, which was deduced to be up to 30-fold and to occur with a half-time of up to 5 s. The results were simulated by a quantitative model, which also explains the varied shapes of cGMP responses to NO found in other cells. Downstream phosphorylation in platelets was detectable within 2 s, and, with continuous exposure (1 min), this pathway could be engaged by subnanomolar NO concentrations (EC 50 ‫؍‬ 0.5 nM).Whereas the spatio-temporal dynamics of some of the primary steps of cellular signal transduction, such as synaptic transmission and changes in intracellular Ca 2ϩ , have become well understood in quantitative terms (1, 2), knowledge of downstream signaling cascades remains largely qualitative. Nevertheless, the kinetic properties of the second messenger cascades are likely to be similarly instrumental in determining how cells respond.A case in point is nitric oxide (NO) 1 signaling. NO acts as a diffusible chemical messenger throughout the body, where it subserves diverse functions, including smooth muscle relaxation, inhibition of platelet aggregation, and the induction of synaptic plasticity (3,4). NO elicits these and other physiological effects by binding to its guanylyl cyclase (GC)-coupled receptors, thereby evoking the accumulation of cGMP in target cells. The receptors are ␣␤-heterodimers, of which two main isoforms, ␣ 1 ␤ 1 and ␣ 2 ␤ 1 , exist at differing levels in different tissues (5). Studies on purified receptors (6) and intact cells (7) suggest that activation of GC occurs very rapidly, within a few ms or less of adding NO, and complete deactivation in cells (upon removal of NO) requires only about 500 ms (7). Furthermore, when studied in isolation, the receptors are highly sensitive NO detectors, with only about 1 nM being needed to evoke half-maximal GC activity (8). The shape of the resulting cGMP response in cells, however, varies greatly from one cell to another. For example,...

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“…2). VASP phosphorylation was determined by Western blot analysis using the anti-phospho(Ser 239 )-VASP antibody 16C2, which detects a 46-kDa VASP phosphorylated on Ser 239 , as well as a 50-kDa VASP phosphorylated on both Ser 239 and Ser 157 (30). The signal for VASP phosphorylation obtained from fresh T cell extracts ( Fig.…”

Section: Cgmp-dependent Protein Kinase In T Cellsmentioning

confidence: 99%

Activation of cGMP-dependent Protein Kinase Iβ Inhibits Interleukin 2 Release and Proliferation of T Cell Receptor-stimulated Human Peripheral T Cells

Fischer

Palmetshofer²,

Gambaryan³

et al. 2001

Journal of Biological Chemistry

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Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK I␤ is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPTcGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK I␤, but not an inactive cGK I␤ mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes.Effective T cell activation can be elicited by a combination of (i) engagement of the T cell antigen receptor (TCR) 1 ⅐CD3complex with foreign antigen presented on major histocompatibility complex molecules and (ii) ligation of the CD28 T cell antigen by B7 molecules (CD80 and CD86) on the surface of antigen-presenting cells, resulting in transcription factor activation, interleukin 2 (IL-2) production, and cell proliferation (1-4). Activation and subsequent proliferation of resting T lymphocytes is an essential process in the T cell-mediated immune response. In vitro, T cells respond to stimulation of the TCR⅐CD3 complex (without costimulation) with IL-2-dependent induction of cell proliferation (2, 5). A costimulatory signal from the CD28 pathway synergizes with the signal from the TCR⅐CD3 complex to further increase cell proliferation and IL-2 production, and T cell proliferation becomes largely independent of IL-2 (6). Intracellular signaling pathways, which may mediate T cell activation, include phosphorylation cascades that stimulate MAP kinases including ERK (3, 7, 8) and p38 kinase (9, 10), as well as phosphatidylinositol 3-kinase (11, 12) and its target Akt/ protein kinase B (13). Reports of cyclic nucleotide effects on T cell activation indicate that cAMP is inhibitory, whereas effects of cGMP have not been conclusively elucidated. In fact, lowering of cAMP levels by induction of phosphodiesterase-7 was reported to be required for T cell activation (14). Nitric oxide, which activ...

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Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

Cited by 312 publications

References 31 publications

Epac inhibits migration and proliferation of human prostate carcinoma cells

Epac inhibits migration and proliferation of human prostate carcinoma cells

Kinetics of a Cellular Nitric Oxide/cGMP/Phosphodiesterase-5 Pathway

Activation of cGMP-dependent Protein Kinase Iβ Inhibits Interleukin 2 Release and Proliferation of T Cell Receptor-stimulated Human Peripheral T Cells

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