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At least 19 recordsLinked to original sources
PubMed1995

Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.

Amino Acid Sequence↗
PubMed1995

Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin.

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+]i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗
PubMed1995

The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins.

BACKGROUND: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. RESULTS: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP. CONCLUSIONS: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.

Actins↗
PubMed2003

A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity.

Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.

3T3 Cells↗
PubMed1992

The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.

Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.

Actin Cytoskeleton↗
PubMed1991

Comparison of vasodilatory prostaglandins with respect to cAMP-mediated phosphorylation of a target substrate in intact human platelets.

The recent purification of a vasodilator-stimulated phosphoprotein (VASP) from human platelets and the development of a specific antiserum against VASP made it possible to study the quantitative effects of cAMP-elevating prostaglandins on cAMP-mediated phosphorylation of VASP in intact human platelets. Prostacyclin (PG-I2), prostaglandin-E1 (PG-E1) and the stable prostacyclinanalog Iloprost, all agents used for the treatment of peripheral vascular disease, induced rapid, stoichiometric and reversible phosphorylation of VASP in human platelets mediated by the cAMP-dependent protein kinase. However, there were substantial differences between these three cAMP-elevating prostaglandins with respect to their effects on extent, duration and reversibility of VASP phosphorylation. Maximal VASP phosphorylation was induced both by PG-I2 and Iloprost, but the PG-I2 effect was only of short duration in comparison to that of Iloprost. The extent of PG-E1-induced VASP phosphorylation was less than that observed with PG-I2 and Iloprost. In endothelial cell-platelet coincubations, an endothelial cell-derived, indomethacin-sensitive factor caused a rapid elevation of platelet cAMP level and VASP phosphorylation. These results provided direct evidence that human endothelial cells are capable of producing biologically active quantities of cAMP-elevating prostaglandins sufficient to induce stoichiometric cAMP-mediated protein phosphorylation in human platelets. VASP-phosphorylation induced by PG-I2 and PG-E1 was completely reversible after removal of the prostaglandins whereas this was only partially the case with Iloprost. In addition, evidence is presented that the prostaglandin-regulated adenylate cyclase system but not the cAMP-mediated protein phosphorylation desensitizes in human platelets after prolonged treatment with cAMP-elevating prostaglandins. VASP phosphorylation is proposed as a marker for quantitating aspects of vessel wall-platelet interaction.

Alprostadil↗
PubMed1992

Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP.

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Analysis, purification and properties of a 50,000-dalton membrane-associated phosphoprotein from human platelets.

Recently, the development of a monospecific antiserum against a 46,000/50,000-dalton membrane protein from human platelets which was stoichiometrically and reversibly phosphorylated in intact human platelets in response to vasodilators was reported. Using this antiserum, the subcellular distribution and the purification of this vasodilator-stimulated phosphoprotein (VASP) from human platelets has now been analysed. The VASP of human platelets is primarily a membrane-associated protein and can be purified to apparent homogeneity by salt extraction and sequential ion-exchange and dye-ligand chromatography with a purification factor of 1200 and a yield of 13%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions indicated that purified monomers of this VASP are linked by interchain disulphide bonding.

Blood Platelets↗
PubMed1992

Inhibition of platelet aggregation by the cAMP-phosphodiesterase inhibitor, cilostamide, may not be associated with activation of cAMP-dependent protein kinase.

We examined the involvement of cAMP-dependent protein kinase (A kinase)2 in the inhibition by cilostamide, a specific inhibitor of the low Km cAMP-phosphodiesterase (PDE), on 9,11-epithio-11,12-methanothromboxane A2 (STA2)-induced platelet aggregation. For comparative purposes, the PGE1 analogue, 17S-20-dimethyl-trans-delta 2-PGE1 (OP-1206) was used. OP-1206 (IC50 = 18 +/- 0.55 nM) and cilostamide (IC50 = 40 +/- 4.5 nM) were both potent inhibitors of the platelet aggregation induced by STA2 (1 microM). OP-1206 and cilostamide dose-dependently inhibited elevations in intracellular free Ca2+ ([Ca2+]i) caused by STA2. OP-1206 caused an almost complete inhibition of Ca2+ mobilization, but cilostamide did not prevent the STA2-induced elevation in [Ca2+]i to the same extent as OP-1206, even at a high concentration (greater than 200 nM). Cilostamide did not increase the cAMP level at concentrations (5-100 nm) which affected STA2-induced aggregation. OP-1206 significantly increased cAMP contents in platelets, and the degree of aggregation inhibition by OP-1206 appears to be related to the size of increase in cAMP. OP-1206 increased phosphorylation of the 50,000 mol. wt vasodilator-stimulated phosphoprotein, at concentrations of 7.9-79 nM, which inhibited aggregation induced by STA2. Cilostamide treatment resulted in a marginal increase in the 50,000 mol. wt phosphorylation at concentrations (10-100 nM) which completely inhibited the STA2-induced aggregation. (8R*, 9S*, 11S*)-(-)-9-Hydroxy-9-n-hexyloxy-8-methyl-2,3,9,10- tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo(a,g)-cycloocta(c,d,e)trinden-1-one (KT-5720), a specific inhibitor of A kinase, not only reversed the inhibition by OP-1206 of STA2-induced platelet aggregation, but also inhibited the OP-1206-induced protein phosphorylation. However, the inhibition by cilostamide of STA2-induced aggregation was not prevented by pretreatment with KT-5720. Inhibition of the STA2-induced aggregation by OP-1206 may be associated with cAMP-dependent protein phosphorylation, while cilostamide may have inhibitory effects on STA2-induced platelet activation through mechanisms other than the activation of A kinase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

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.

Cell Extracts↗
PubMed1995

Synergistic phosphorylation of platelet rap1B by SIN-1 and iloprost.

Human platelets suspended in plasma or buffer were incubated with low concentrations of the nitric oxide (NO)-donor 3-morpholino-syndnonime (SIN-1; 100 nM to 1 microM) and the stable prostacyclin analogue iloprost (50 or 100 pM) and analyzed for cyclic nucleotide levels and protein phosphorylation. SIN-1 and iloprost synergistically stimulated the phosphorylation of rap1B and the 50 kDa vasodilator-stimulated phosphoprotein. SIN-1 stimulated platelet cyclic GMP and cAMP-levels and enhanced the increase in cyclic AMP elicited by iloprost. It was found that the mechanism underlying the synergistic phosphorylation of the 50 kDa protein and rap1B was different: synergistic phosphorylation of the 50 kDa protein seemed to be mediated by activation of both protein kinases A and G, whereas the synergistic rap1B phosphorylation could be attributed entirely to activation of protein kinase A. Measurement of rap1B phosphorylation might be a useful tool to monitor the action of systemically applied prostacyclin-analogues and nitrovasodilators in pharmacological studies.

Autoradiography↗
PubMed1991

Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells.

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

Animals↗
PubMed1995

Platelet rap1B phosphorylation is a sensitive marker for the action of cyclic AMP- and cyclic GMP-increasing platelet inhibitors and vasodilators.

Rap1B, a ras-like protein expressed in high concentrations in human platelets, serves as a substrate for protein kinase A (PKA) and, eventually, protein kinase G (PKG). We measured rap1B phosphorylation by autoradiography of 32P-labeled proteins in platelets prelabeled with [32P]-orthophosphate. Platelets coincubated with histamine-stimulated human umbilical vein endothelial cells (EC) showed increased phosphorylation of the 50-Kd vasodilator-stimulated phosphoprotein (VASP) of 2.6 +/- 0.5-fold maximally and of rap1B of 17.5 +/- 7.1-fold maximally (mean +/- SE, n = 4). Incubation of platelets with prostacyclin (PGI2), the PGI2-analogue iloprost (ILO), the nitric oxide (NO) donors SIN-1 or sodium nitroprusside (SNP) showed greater concentration-dependent phosphorylation of rap1B than of VASP. Phosphorylation of rap1B had a slow time course and was irreversible in contrast to that of VASP, which was rapid and reversible. Phosphorylation of rap1B was dependent on an increase of platelet cyclic AMP and/or cyclic GMP. Very small concentrations of ILO (50 pM), PGI2 (1 nM), and SIN-1 (100 nM) increased rap1B phosphorylation. Rap1B phosphorylation could also be detected by Western blot after incubation of platelet-rich plasma (PRP) with ILO or SIN-1. Measurement of platelet rap1B phosphorylation is a novel tool that allows monitoring of the action of labile (PGI2, NO) and more stable (ILO, SIN-1, SNP) platelet inhibitors and vasodilators that increase intracellular cyclic AMP and cyclic GMP. Determination of rap1B phosphorylation by Western blot opens new possibilities of measuring platelet-EC interactions in clinical studies and of monitoring the action of systemically applied PGI2 analogues and nitrovasodilators in pharmacologic studies.

Arginine↗
PubMed1992

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)

Adult↗
PubMed1991

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., Schanzenbächer, 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.

Autoradiography↗
PubMed1990

Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators.

Recently, we reported the purification of a 46-kDa membrane-associated platelet protein which is phosphorylated in intact platelets and platelet membranes by cGMP- and cAMP-dependent protein kinases (Halbrügge, M., and Walter, U. (1989) Eur. J. Biochem. 185, 41-50). Here we demonstrate that both cGMP- and cAMP-dependent protein kinases catalyze the rapid incorporation of up to 1.4 mol of phosphate/mol of this purified vasodilator-stimulated phosphoprotein (VASP). A specific rabbit antiserum was prepared which recognized both the 46-kDa dephospho form and the 50-kDa phospho form of VASP in Western blots. In untreated washed platelets, VASP was found to be present primarily as a 46-kDa dephosphoprotein. Sodium nitroprusside (100 microM) raised the intracellular platelet cGMP concentration from approximately 0.44 to 4.1 microM, without a significant effect on the cAMP level, and converted up to 50% of VASP to the 50-kDa phospho form. Prostaglandin E1 (10 microM) raised the platelet cAMP concentration from approximately 4.4 to 28.4 microM, without a significant effect on the cGMP level, and shifted up to 67% of VASP to the 50-kDa phospho form. Removal of the vasodilators sodium nitroprusside and prostaglandin E1 from the platelet suspension was followed by a return of the cyclic nucleotide concentration to basal levels and subsequent conversion of the 50-kDa phospho form of VASP to the 46-kDa dephospho form. The results support the hypothesis that VASP phosphorylation is an important component of the intracellular mechanism of action of these vasodilators in human platelets.

Alprostadil↗
PubMed2026

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of capping protein and VASP.

Cell migration frequently employs protrusions termed lamellipodia, constituting the prime model system for generation of branched actin filament networks. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and Ena/VASP family polymerases in lamellipodial actin assembly. We show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. While Pfn promotes Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal vanishes in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, differentially regulating VASP and its antagonizer CP. Mathematical modeling of our data suggest Ena/VASP and CP to compete for recruitment to lamellipodial edges. Our work provides critical insights into the molecular logic of branched actin network assembly in protrusion and force generation.

Profilins↗