The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.     

Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I

cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Ibeta mutant (cGK Ibeta-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Ibeta-K405A, caused a 1.5-2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.     

Protein phosphorylation regulated by cyclic nucleotide-dependent protein kinases in cell extracts and in intact human lymphocytes.

A specific 46,000/50,000 molecular weight protein substrate for both cAMP-dependent protein kinase (cAK) and cGMP-dependent protein kinase (cGK) extensively characterized and purified from human platelets was found to be present also in human T-lymphocytes, B-lymphocytes and other cells and tumour cell lines. This protein termed vasodilator-stimulated phosphoprotein (VASP) was present in cytosol and membranes of lymphocytes. Addition of exogenous purified cAK or cGK to lymphocyte cytosol or membranes converted 80-90% of VASP to its phosphoform. Endogenous VASP phosphorylation in both cytosol and membranes was stimulated by the addition of cAMP but not by cGMP. With intact lymphocytes, prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) induced an increase of cAMP and converted 70% of VASP to its phosphoform. In contrast, an increase of cGMP was not associated with VASP phosphorylation although cGK was detected in lymphocytes. These data support the hypothesis that VASP phosphorylation may be an important component of cAMP-mediated regulation of lymphocyte function.     

Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells.

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Signal pathway responsible for hepatocyte preconditioning by nitric oxide

Nitric oxide (NO) improves liver resistance to hypoxia/reperfusion injury acting as a mediator of hepatic preconditioning. However, the mechanisms involved are still poorly understood. In this study, we have investigated the mechanisms by which short-term exposure to the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium-1,2-diolate (NOC-9) increases hepatocyte tolerance to hypoxic injury. Isolated rat hepatocytes preincubated 15 min with NOC-9 (0.250 mM) became resistant to the killing caused by hypoxia. NOC-9 cytoprotection did not involve the activation of protein kinase C, but was instead blocked by inhibiting soluble guanylate cyclase with 1H-(1,2,4)-oxadiazolo-(4,3) quinoxalin-1-one (ODQ) (50 microM) or cGMP-dependent kinase (cGK) with KT 5823 (5 microM). Conversely, cGMP analogue, 8Br-cGMP (50 microM) mimicked the effect of NOC-9. Western blot analysis revealed that hepatocyte treatment with NOC-9 or 8Br-cGMP significantly increased dual phosphorylation of p38 MAPK. The activation of p38 MAPK was abolished by inhibiting guanylate cyclase or cGK. Pretreatment with NO significantly reduced intracellular Na(+) accumulation in hypoxic hepatocytes. This effect was reverted by KT 5823 as well as by the p38 MAPK inhibitor SB203580. SB203580 also reverted NOC-9 protection against hypoxic injury. Altogether, these results demonstrated that NO can induce hepatic preconditioning by activating p38 MAPK through a guanylate cyclase/cGK-mediated pathway.     

Inhibition of cGMP-dependent protein kinases potently decreases neutrophil spontaneous apoptosis

The signalling pathways mediating neutrophil spontaneous apoptosis are still largely unknown. We report that the indolocarbazole compound KT5823, a specific inhibitor of cGMP-dependent protein kinases (cGK), dose-dependently inhibited spontaneous apoptosis of neutrophils. At the concentration eliciting the maximum effect (8 microM), it decreased apoptosis from 72.42+/-12.79% to 45.86+/-7.22% (p=0.0002, n=6). Similarly, the isoquinoline sulfonamide compound H89, another cGK inhibitor, prevented neutrophil apoptosis. At the concentration eliciting the maximum effect (20 microM), it decreased apoptosis from 72.42+/-12.79% to 31.84+/-10.70% (p=0.0004, n=6). The maximum effect of KT5823 and H89 was comparable to that of GM-CSF and LPS, respectively. Moreover, YC-1, a soluble guanylate cyclase activator, and 4-([3',4',-(methylenedioxy)benzyl]amino)-6-methoxyquinazoline, a specific phosphodiesterase 5 inhibitor, enhanced neutrophil apoptosis, and their effect was antagonised by KT5823. Taken together, these observations highlight a new role of cGK as important mediators of neutrophil spontaneous apoptosis.     

Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA

The role of RhoA in myosin light-chain (MLC)(20) dephosphorylation and smooth muscle relaxation by PKA and PKG was examined in freshly dispersed and cultured smooth muscle cells expressing wild-type RhoA, constitutively active Rho(V14), and phosphorylation site-deficient Rho(A188). Activators of PKA (5,6-dichloro-1-beta-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothionate, Sp-isomer; cBIMPS) or PKG [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG (VIP) induced phosphorylation of constitutively active Rho(V14) and agonist (ACh)- or GTPgammaS-stimulated wild-type RhoA but not Rho(A188). Phosphorylation was accompanied by translocation of membrane-bound wild-type RhoA and Rho(V14) to the cytosol and complete inhibition of ACh-stimulated Rho kinase and phospholipase D activities, RhoA/Rho kinase association, MLC(20) phosphorylation, and sustained muscle contraction. Each of these events was blocked depending on the agent used, by the PKG inhibitor KT5823 or the PKA inhibitor myristoylated PKI. Inhibitors were used at a concentration (1 microM) previously shown by direct measurement of kinase activity to selectively inhibit the corresponding kinase. In muscle cells overexpressing the active phosphorylation site-deficient mutant Rho(A188), MLC(20) phosphorylation was partly inhibited by SNP, VIP, cBIMPS, and 8-pCPT-cGMP, suggesting the existence of an independent inhibitory mechanism downstream of RhoA. Results demonstrate that dephosphorylation of MLC(20) and smooth muscle relaxation are preferentially mediated by PKG- and PKA-dependent phosphorylation and inactivation of RhoA.     

Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells

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 Ibeta 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-pCPT-cGMP, 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 Ibeta, but not an inactive cGK Ibeta 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.     

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation,platelet aggregation,and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.     

PDE5 inhibitor promotes melanin synthesis through the PKG pathway in B16 melanoma cells

PDE inhibitors could increase cellular cGMP levels and are used to treat erectile dysfunction as well as pulmonary arterial hypertension. cGMP production was reported to be necessary for UVB-induced melanin synthesis, however, the effect of PDE5 inhibitor on melanin synthesis has not been examined. We found that PDE5 inhibitor (sildenafil or vardenafil) and the cGMP analog 8-CPT-cGMP stimulated CREB phosphorylation, leading to increased tyrosinase expression and melanin synthesis, which was counteracted by KT5823, a selective cGMP-dependent protein kinase (PKG) inhibitor. However, KT5823 did not affect cAMP-elevating agent-mediated melanin synthesis, indicating that KT5823 selectively inhibited cGMP-induced melanin synthesis. This is the first study to find that PDE5 inhibitor can promote melanin synthesis and reveal that PKG-dependent CREB phosphorylation and tyrosinase expression is involved in cGMP-induced melanin synthesis. Our results suggest that PDE5 inhibitor may be beneficial for the treatment of hypopigmentation diseases.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.     

Effect of cGMP analogues and protein kinase G blocker on secretory activity, apoptosis and the cAMP/protein kinase A system in porcine ovarian granulosa cells in vitro

The aim of the present study was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian functions. In the first series of experiments we studied the effects of the cGMP analogues 8-pCPT-cGMP (0.001-100 nM), Rp-8-pCPT-cGMPS (0. 01-100 nM), Rp-8-Br-cGMPS (0.01-100 nM), and Rp-8-Br-PET-cGMPS (0.01-100 nM) on the release of progesterone, insulin-like growth factor I (IGF-I) and oxytocin by cultured porcine granulosa cells. In a second series of experiments, the effects of Rp-8-Br-PET-cGMPS (50 nM) and KT5822 (100 ng/ml), specific inhibitor of cGMP-dependent protein kinase (PKG), on cAMP, PKA, oxytocin and the occurrence of apoptosis in cultured cells were compared. The release of hormones and IGF-I into the culture medium was evaluated using a RIA, while the percentage of cells containing visible oxytocin, cAMP, as well as the regulatory and catalytic subunits of PKA was assessed using immunocytochemistry. Occurrence of apoptosis in these cells was detected using the TUNEL method. The stimulatory (8-pCPT-cGMP and Rp-8-pCPT-cGMPS), inhibitory (Rp-8-Br-cGMPS) and biphasic (Rp-8-Br-PET-cGMPS) effect of cGMP analogues on progesterone release was observed. All cGMP analogues used suppressed IGF-I release. All cGMP analogues decreased oxytocin release, but 8-pCPT-cGMP and Rp-8-Br-cGMPS, when given at low doses (0.01-0.1 and 1-10 nM, respectively) stimulated oxytocin output. Both, Rp-8-Br-PET-cGMPS and KT5822 increased the rate of incidence of apoptosis and percentage of cells containing immunoreactive cAMP. Both Rp-8-Br-PET-cGMPS and KT5822 decreased the proportion of cells containing immunoreactive oxytocin and regulatory subunit of PAK KT5822, but not Rp-8-Br-PET-cGMPS, increased the number of cells containing catalytic subunit of PKA. The present observations suggest the involvement of cGMP and PKG in control of the production of steroid, nonapeptide hormone, growth factor, cAMP and cAMP-dependent PKA, as well as the induction of apoptosis in porcine ovarian cells.     

Cyclic GMP induced apoptosis via protein kinase G in oestrogen receptor-positive and -negative breast cancer cell lines

The activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. The present study was designed to examine the effects of cGMP and PKG on cell growth and apoptosis in the human breast cancer cell lines, MCF-7 and MDA-MB-468. To achieve this, 1-benzyl-3-(5P-hydroxymethyl-2P-furyl) indazole (YC-1), a soluble guanylyl cyclase activator, and 8-bromo-cGMP (8-br-cGMP), a membrane-permeant and phosphodiesterase-resistant analogue of cGMP, were employed in MCF-7 and MDA-MB-468 cells. Then, the role of PKG in the induction of apoptosis was evaluated using KT5823 and Rp-8-pCPT-cGMP as specific inhibitors of PKG. The expression of PKG isoforms in these cell lines was also investigated. KT5823 and Rp-8-pCPT-cGMP significantly attenuated the loss of cell viability caused by YC-1 and 8-br-cGMP in these cells. This study provides direct evidence that the activation of PKG by cGMP induces growth inhibition and apoptosis in MCF-7 and MDA-MB-468 breast cancer cell lines.     

Progesterone regulates granulosa cell viability through a protein kinase G-dependent mechanism that may involve 14-3-3sigma

Progesterone (P4) inhibits granulosa cell and spontaneously immortalized granulosa cell (SIGC) apoptosis by regulating membrane-initiated events. However, the nature of the signal transduction pathway that is induced by these membrane-initiated events has not been defined. To gain insights into the P4-regulated signal transduction pathway, mouse granulosa cells and SIGCs were cultured with 8-br-cGMP and P4. In culture, 8-br-cGMP mimicked P4's antiapoptotic actions. Because cGMP activates protein kinase G (PKG), the effect of PKG antagonists on P4-regulated SIGC viability was assessed. P4's antiapoptotic action was attenuated by the PKG inhibitors, Rp-8-pCPT-cGMP, KT5823, the PKG-1alpha-specific inhibitor, DT-3, and a dominant negative PKG-1alpha. Further, the type I isoform of PKG was shown to be expressed by SIGCs and activated by P4. P4's antiapoptotic action was not affected by the PKA inhibitor, KT5720. Collectively, these findings indicate that P4 maintains SIGC viability by activating PKG-1alpha. PKG-1alpha-GFP was shown to localize predominantly to the cytoplasm of SIGCs. To identify potential cytoplasmic targets of PKG-1alpha, SIGCs were cultured for 5 h with P4 in the presence or absence of DT-3. Cell lysates were prepared and subjected to two-dimensional electrophoresis. The resulting gels were sequentially stained with ProQ-Diamond Gel Stain and Coomassie Blue to reveal phosphorylated proteins. The two-dimensional gels revealed one major protein, the phosphorylation status of which was abrogated by DT-3. Mass spectrometric analysis identified this protein as 14-3-3sigma, with 14-3-3sigma being phosphorylated on tyrosine 19, serine 28, serine 69, serine 74, threonine 90, threonine 98, and serine 116. Finally, difopein, a specific 14-3-3 inhibitor, was shown to induce apoptosis even in the presence of serum. These data suggest that 1) P4 regulates the phosphorylation status of 14-3-3sigma through a PKG-dependent pathway and 2) 14-3-3sigma plays a central and essential role in maintaining the viability of SIGCs.     

3',5'-Cyclic Guanosine Monophosphate Activates Mitogen-Activated Protein Kinase in Rat Pinealocytes

The role of 3',5'-cyclic guanosine monophosphate (cGMP) in the activation of mitogen-activated protein kinases (MAPKs) was investigated in rat pinealocytes. Treatment with dibutyryl cGMP (DBcGMP) dose-dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of DBcGMP was abolished by PD98059 (a MAPK kinase inhibitor), H7 (a nonspecific protein kinase inhibitor), and KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor]. Elevation of cellular cGMP content by treatment with norepinephrine, zaprinast (a cGMP phosphodiesterase inhibitor), or nitroprusside was effective in activating MAPK. Natriuretic peptides that were effective in elevating cGMP levels in this tissue were also effective in activating MAPK. Our results indicate that, in this neuroendocrine tissue, the cGMP/PKG signaling pathway is an important mechanism used by hormones and neurotransmitters in activating MAPK.     

A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.     

Regulation of the L-Type Ca2+ Channel Current in Rat Pinealocytes

Abstract : In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca²⁺ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca²⁺ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.     

Endothelial cell-dependent phosphorylation of a platelet protein mediated by cAMP- and cGMP-elevating factors.

We reported previously that a 46/50-kDa membrane-associated vasodilator-stimulated phosphoprotein (VASP) is phosphorylated in intact human platelets in response to both cGMP- and cAMP-elevating vasodilator drugs and presented evidence that this is mediated by cGMP- and cAMP-dependent protein kinases, respectively. VASP was recently purified and an antibody against it was developed which detects a phosphorylation-induced mobility change of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Halbrügge, M., Friedrich, C., Eigenthaler, M., Schanzenb?cher, P., and Walter, U. (1990) J. Biol. Chem. 265, 3088-3093). We have now used these methods for the quantitative analysis of VASP phosphorylation during coincubations of human endothelial cells and human platelets. Endothelial cell-derived factors caused the rapid, stoichiometric, and reversible phosphorylation of platelet VASP during these coincubations. Other experiments indicated that the endothelium-derived factors which stimulate VASP phosphorylation are prostacyclin and endothelium-derived relaxing factor whose effects are mediated by cAMP/cAMP-dependent protein kinase and cGMP/cGMP-dependent protein kinase, respectively. The results suggest that VASP phosphorylation is an important component of the inhibitory effects of prostacyclin and endothelium-derived relaxing factor on platelet activation and that VASP phosphorylation is a useful biochemical marker for the interaction of endothelial cells and platelets.     

VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation

Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway.     

PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.