Phosphorylation Of Rap1b Research Articles

Host Cell Rap1b mediates cAMP-dependent invasion by Trypanosoma cruzi.

Trypanosoma cruzi cAMP-mediated invasion has long been described, however, the detailed mechanism of action of the pathway activated by this cyclic nucleotide still remains unknown. We have recently demonstrated a crucial role for Epac in the cAMP-mediated invasion of the host cell. In this work, we gathered evidence indicating that the cAMP/Epac pathway is activated in different cells lines. In accordance, data collected from pull-down experiments designed to identify only the active form of Rap1b (Rap1b-GTP), and infection assays using cells transfected with a constitutively active mutant of Rap1b (Rap1b-G12V), strongly suggest the participation of Rap1b as mediator of the pathway. In addition to the activation of this small GTPase, fluorescence microscopy allowed us to demonstrate the relocalization of Rap1b to the entry site of the parasite. Moreover, phospho-mimetic and non-phosphorylable mutants of Rap1b were used to demonstrate a PKA-dependent antagonistic effect on the pathway, by phosphorylation of Rap1b, and potentially of Epac. Finally, Western Blot analysis was used to determine the involvement of the MEK/ERK signalling downstream of cAMP/Epac/Rap1b-mediated invasion.

Phosphorylation-induced Conformational Changes in Rap1b: ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.

CAMP-dependent Oncogenic Action of Rap1b in the Thyroid Gland

cAMP signaling leads to activation and phosphorylation of Rap1b. Using cellular models where cAMP stimulates cell proliferation, we have demonstrated that cAMP-mediated activation, as well as phosphorylation of Rap1b, is critical for cAMP stimulation of DNA synthesis. To determine whether Rap1b stimulates mitogenesis in vivo, we have constructed a transgenic mouse where a constitutively active G12V-Rap1b, flanked by Cre recombinase LoxP sites, is followed by the dominant negative S17N mutant. Employing this novel mouse model, we have switched, in a tissue-specific (thyroid) and temporally controlled manner, the expression of Rap1b from a stimulatory to an inhibitory form. These experiments provide conclusive evidence that Rap1b is oncogenic in the thyroid in ways linked to transduction of the cAMP mitogenic signal.

CAMP Inhibition of Akt Is Mediated by Activated and Phosphorylated Rap1b

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.

Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology.

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein-protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.

Nitric Oxide Stimulates the Phosphorylation of rap1b in Human Platelets and Acts Synergistically with Iloprost

Phosphorylation of rap1b in human platelets correlates with both an upward shift of the protein on sodium dodecyl sulfate polyacrylamide gels and the translocation of the phosphorylated protein to the cytosolic fraction of platelets. We reported that this phenomenon occurs in platelets in response to agents that stimulate adenylate cyclase and thereby activate the cyclic AMP-dependent protein kinase. We now have evidence that phosphorylation of rap1b in platelets is also induced by nitric oxide generating compounds through stimulation of guanylate cyclase and activation of the cyclic GMP-dependent protein kinase. We observed time-dependent phosphorylation of rap1b and dose-dependent inhibition of collagen-stimulated aggregation in washed platelets incubated with S-nitroso serum albumin. In the presence of a combination of iloprost and 3-morpholino-sydnonimine, when both PKA and PKG are activated, phosphorylation of rap1b increased synergistically to a level three times higher than the sum of their individual actions.

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.

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-sydnonime (SIN-1; 100 nM to 1 μM) 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.

Formation of biologically active autacoids is regulated by calcium influx in endothelial cells.

The blocker of receptor-mediated calcium entry SK&F 96365 was used to evaluate the contribution of calcium influx to the formation of biologically active endothelial prostanoids and endothelium-derived relaxing factor (EDRF). SK&F 96365 inhibited histamine-stimulated calcium entry into human umbilical vein endothelial cells but not its discharge from intracellular stores as determined spectrofluorometrically by changes of intracellular calcium concentration in fura-2-loaded cells. Concordantly, SK&F 96365 inhibited histamine-induced endothelial synthesis of 6-keto-prostaglandin F1 alpha and thromboxane B2 in a dose-dependent manner. To assess the functional significance of endothelial formation of prostacyclin and EDRF to platelets, the cAMP- and cGMP-dependent phosphorylation of two platelet proteins, rap1B and a 50-kD vasodilator-stimulated phosphoprotein (VASP), was analyzed in coincubation experiments of endothelial cells with platelets. Autacoids released by histamine-stimulated endothelial cells caused the phosphorylation of rap1B and VASP in platelets, which was only partly inhibited by either indomethacin or NG-monomethyl-L-arginine but was almost completely suppressed by SK&F 96365. The concomitant endothelial release of thromboxane A2 had no effect on protein kinase C- and calcium-dependent phosphorylation of platelet proteins. The results demonstrate that blockade of receptor-mediated calcium entry by SK&F 96365 markedly reduced the release of biologically active prostacyclin and EDRF from endothelial cells. Thus, calcium influx but not calcium release from intracellular stores plays a critical role in the receptor-stimulated formation and liberation of prostacyclin and EDRF in endothelial cells.

Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca2+-ATPase and Rap1B in platelets and various cell lines

Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylation of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. As this transport was recently found to be controlled by two isoforms of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA), the 100 kDa SERCA2b and the newly identified 97 kDa SERCA, we attempted to establish which isoform is involved in this regulation. For this purpose, we studied the expression and regulation of both the SERCA and Rap1 isoforms in platelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determined by detection of its phosphoenzyme formation and by Western blotting using an isoform-specific antibody. In contrast, the expression of the 97 kDa SERCA, studied by the same methods, varied from total absence in the cancer cells to high levels in the megakaryocytic cell lines. With regard to the potential regulatory Rap1 proteins, Western blotting showed different expression of total Rap1 isoforms among the cell lineages, thus ruling out any possible relationship between Rap1 and SERCA2b. However, the expression of Rap1 proteins correlated with that of the 97 kDa SERCA isoform. More refined analysis of the rap1A and rap1B isoforms by reverse transcription PCR and by determining cAMP-induced phosphorylation of Rap1B, i.e. its functional mechanism, confirmed the correlation between Rap1B and the 97 kDa SERCA expression. This relationship was also established by the concerted up-regulation of these two proteins demonstrated in the pathological model of platelets from hypertensive rats. It is concluded that the expressions of 97 KDa SERCA and Rap1B are related, suggesting that regulation of the platelet Ca(2+)-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.

Effect of cAMP on the association of small GTP-binding proteins with the cytoskeleton of human platelets

Following activation of human platelets changes in cytoskeletal organization occur: some proteins, which are present in the cytosol or membrane-associated in resting platelets, are recovered in the Triton-insoluble residue in activated cells. Assembly and disassembly of complex effector units on the membrane and inside cells is under the control of low molecular weight GTP-binding proteins, particularly those in the ras family. We investigated the interaction of small GTP-binding proteins with the platelet cytoskeleton and the effect of high cAMP levels on these interactions. At least two GTP-binding proteins of 24 h and 28 kDa were detected in the Triton-insoluble residue of resting platelets. Stimulation of platelets with thrombin or concanavalin A (Con A), under non-aggregating conditions, resulted in increased 24 kDa protein-bound GTP, which also contained a significant amount of rap1B. High cAMP levels differently affected this interaction depending on the type of agonist used. cAMP increased association of G-proteins with the cytoskeleton following Con A-activation, while it decreased G-proteins interaction after thrombin stimulation. The activation did not influence the cAMP-dependent phosphorylation of rap1B. No phosphoprotein corresponding to rap1B could be detected in the Triton-insoluble residues, however. These findings could be related to the different mechanims of cytoskeletal protein recruitment in platelets activated with either thrombin or Con A.

Mutational analysis of the cAMP-dependent protein kinase-mediated phosphorylation site of Rap1b.

Rap1b, a member of the Ras superfamily of low molecular weight GTP-binding proteins, can be phosphorylated by cAMP-dependent protein kinase (protein kinase A). The experiments presented here were undertaken to determine the precise site of this phosphorylation. Because the Rap1 proteins are highly homologous, there are no specific antibodies able to discriminate between them. To overcome this problem, we used a transient expression system of a fused protein containing in the NH2 terminus an epitope for a known antibody. Using this system, the transfected protein was expressed at a high level and was localized in a perinuclear structure, as previously reported for the endogenous Rap1 proteins. The mutational analysis of Rap1b revealed Ser179 as the residue involved in the protein kinase A-mediated phosphorylation. The presence of a Lys179 instead of the wild-type Ser179 (resembling the Rap1a sequence) rendered Ser180 a better substrate for phosphorylation caused by protein kinase A. The mobility shift of Rap1b in SDS gels, observed in cells that were stimulated with agonists that increase cAMP, was caused, at least in part, by the phosphorylation of Rap1b.

Phosphorylation of rap1B by protein kinase a is not involved in platelet inhibition by cyclic AMP

The functional consequence of cyclic AMP-dependent phosphorylation of rap1B for stimulus-induced platelet activation is not known. Platelets were pretreated with the stable prostacyclin-analogue iloprost and resuspended in plasma without iloprost. Western blot analysis showed that rap1B was completely converted into its phosphorylated form in the iloprost-pretreated platelets. Surprisingly, the platelets that contained phosphorylated rap1B were found to respond fully to activation by a wide variety of stimuli: aggregation upon stimulation by collagen, phorbol ester, vasopressin, ADP, epinephrine, and ATP-secretion from dense granules induced by collagen, thrombin-receptor activating peptide, vasopressin and phorbol ester were unchanged as compared to control. The results indicate that cyclic AMP-dependent phosphorylation of rap1B does not play a role in the inhibition of the various signal transduction pathways that lead to platelet aggregation and dense granule secretion.

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.

Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition

Exposure of human platelets to prostacyclin (PGI2), iloprost or prostaglandin E1 (PGE1) elicits the cyclic AMP-dependent phosphorylation of proteins of 22, 24, 30, 39, 50, 60 and 250 kDa (P22, P24 etc.). P22 was recently identified as rap 1B, a ras-like protein, and P24 was shown to be the beta-chain of glycoprotein Ib. We found that cyclic AMP-dependent phosphorylation of all proteins except P22 was maximal 1 min after exposure of platelets to PGI2, iloprost or PGE1; maximal phosphorylation of P22 occurred after 45 min of incubation. Inhibition of thrombin-induced platelet activation required only a 30 s incubation with PGI2 or iloprost; at this time phosphorylation of P22 was only slightly increased. Although at maximal concentrations PGI2 was more potent than PGE1 in inhibiting thrombin-induced platelet activation, no difference in the degree and the kinetics of cyclic AMP-dependent protein phosphorylation was found. Platelets that had been preincubated and washed in the presence of PGE1 and later resuspended in the absence of PGE1 responded fully to activation by thrombin despite maximal phosphorylation of P22 and P24. Furthermore, addition of PGI2 to PGE1-washed platelets prevented thrombin-induced platelet activation, but did not evoke further phosphorylation of P22 or P24. Phosphorylation of P39 and P50 correlated better with PGI2-induced inhibition of platelet activation. In experiments in which PGE1-induced inhibition of platelet activation was overcome by the addition of thrombin, no dephosphorylation of proteins phosphorylated by cyclic AMP-dependent kinases was observed. These experiments indicate that: (a) phosphorylation of rap 1B and glycoprotein Ib is not related to platelet inhibition by cyclic AMP; (b) phosphorylation of other proteins such as P39 and P50 probably plays a role in mediating cyclic AMP-dependent platelet inhibition; (c) reactions other than cyclic AMP-dependent protein phosphorylation may participate in platelet inhibition by cyclic AMP.