Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RasGRF1 is a member of the Ras guanine nucleotide exchange factor (RasGEF) family of proteins which are directly responsible for the activation of Ras and Rac GTPases. Originally identified as a phosphoprotein, RasGRF1 has been shown to be phosphorylated by protein kinase A and more recently, by the non-receptor tyrosine kinases Ack1 and Src. In this report we show that RasGRF1 interacts with and is phosphorylated by Cdk5 on serine 731 to regulate its steady state levels in mammalian cells as well as in neurons. Phosphorylation on this site by Cdk5 leads to RasGRF1 degradation through a calpain-dependent mechanism. Additionally, cortical neurons from Cdk5 knockout mice have higher levels of RasGRF1 which are reduced when wild-type Cdk5 is transfected into these neurons. In mitotic cells, nuclei become disorganized when RasGRF1 is overexpressed and this is rescued when RasGRF1 is co-expressed with active Cdk5. When RasGRF1 levels are elevated in neurons through overexpression of either the wild-type RasGRF1, or the phosphorylation mutant of RasGRF1 and by the transfection of a dominant negative Cdk5 construct, nuclei appeared condensed and fragmented. On the other hand, a reduction of RasGRF1 levels through p35/Cdk5 overexpression also leads to nuclear condensation in neurons. These data show that phosphorylation of RasGRF1 by Cdk5 tightly regulates its levels, which is essential for proper cellular organization.
 ...
PMID:Neuronal nuclear organization is controlled by cyclin-dependent kinase 5 phosphorylation of Ras Guanine nucleotide releasing factor-1. 1692 Dec 54

Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of G(s)alpha protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of G(s)alpha/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.
 ...
PMID:A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation. 1700 Jul 74

Cyclic adenosine 3',5'-monophosphate (cAMP) signaling regulates the expression of fertilizing ability in mammalian spermatozoa. Many articles indicate that this signaling is mediated mainly via protein kinase A. Recently, a guanine nucleotide exchange factor for small G protein Rap1 (an exchange protein directly activated by cAMP: Epac) was discovered as a new mediator of cAMP signaling in somatic cells. The aim of this study was to reveal the existence of cAMP-Epac signaling in mouse spermatozoa. Northern blot analysis and in situ hybridization suggested that Epac1 and Epac2 mRNAs were transcribed in the seminiferous epithelia of the testis. This shows that expression of Epac mRNAs is present in mouse testicular germ cells. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. Moreover, treatment of epididymal spermatozoa with an Epac-specific cAMP analog, 8-pMeOPT-2'-O-Me-cAMP, induced activation of Rap1, as revealed with a commercial kit for pull-down assay. These results indicate the existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.
 ...
PMID:Evidence for existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa. 1707 80

PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.
 ...
PMID:Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function. 1722 22

Nuclear factor-kappa B (NF-kappaB) is regulated by various stimulants to show many physiological results. Lipopolysaccharide (LPS) activates NF-kappaB through toll-like receptor 4 (TLR4)-dependent signal transduction. LPS-treatment also produces cyclic AMP (cAMP) in Raw 264.7 murine macrophages. Two principal effector proteins for cAMP are protein kinase A (PKA) and cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Here, we investigated whether NF-kappaB can be activated by cAMP production through Epac1-mediated Rap1 activation by using Epac-specific cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT). NF-kappaB activity was increased by the treatment with CPT but it was reduced by co-transfection with dominant negative of Rap1 (Rap1N17). In conclusion, NF-kappaB activation should be regulated through Epac1-mediated Rap1 stimulation for LPS-induced inflammatory responses in murine macrophages. It suggests that Epac1-mediated Rap1/NF-kappaB pathway could be helpful for interpretation on various cAMP-mediated physiological responses and it could be used as a target to control their pathological abnormalities.
 ...
PMID:Lipopolysaccharide stimulates Epac1-mediated Rap1/NF-kappaB pathway in Raw 264.7 murine macrophages. 1753 77

A critical process in angiogenesis is endothelial cell proliferation, which requires activation of extracellular signal-regulated kinase (ERK)1/2. This study analyzed the pathway responsible for adenosine-induced ERK1/2 phosphorylation in human umbilical vein endothelial cells (HUVEC). Characterization with adenosine receptor (AR) agonists and antagonists and the AR mRNA profile demonstrated that stimulation of the A(2B)AR can mediate ERK1/2 phosphorylation in HUVEC. The lack of sensitivity of A(2B)AR-mediated ERK1/2 phosphorylation to 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and 3-[1-[3-(amidinothio)propyl]-1H-in-dol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro31-8220) indicated that protein kinase C stimulation is not required. The response did not involve transactivation of receptors for epidermal growth factor or vascular endothelial growth factor (VEGF). The A(2B)AR-mediated response required functional G(alphas) and was mimicked by forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate. However, ERK1/2 phosphorylation induced by A(2B)AR stimulation and forskolin was insensitive to protein kinase A inhibitors. It was hypothesized that the A(2B)AR-mediated ERK1/2 activation may involve exchange protein activated by cAMP (Epac), a cAMP-activated guanine nucleotide exchange factor for Rap GTPases. Reverse Transcription-polymerase chain reaction analysis detected Epac1 but not Epac2 in HUVEC. 8-(p-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), an Epac activator, stimulated ERK1/2 phosphorylation. Overexpression of Epac1 enhanced A(2B)AR-mediated and forskolin-induced ERK1/2 phosphorylation, whereas response to VEGF was unaffected. Inhibition of Epac1 expression with small interfering RNA substantially reduced A(2B)AR-mediated and forskolin-induced ERK1/2 phosphorylation and abolished that by 8CPT-2Me-cAMP. A(2B)AR stimulation and forskolin activated Rap1. Expression of a dominant-negative Ras protein did not affect either forskolin-induced or A(2B)AR-mediated ERK1/2 phosphorylation. In summary, Epac1 activation in HUVEC results in ERK1/2 activation, and this protein, at least in part, mediates response to the physiologically relevant event of A(2B)AR stimulation.
 ...
PMID:Cyclic AMP-dependent, protein kinase A-independent activation of extracellular signal-regulated kinase 1/2 following adenosine receptor stimulation in human umbilical vein endothelial cells: role of exchange protein activated by cAMP 1 (Epac1). 1756 9

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.
 ...
PMID:Specificity and mechanism of action of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases. 1793 39

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.
 ...
PMID:Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states. 1797 84

Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.
 ...
PMID:Epac/Rap and PKA are novel mechanisms of ANP-induced Rac-mediated pulmonary endothelial barrier protection. 1806 50

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity.
 ...
PMID:PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells. 1808 13


<< Previous 1 2 3 4 5 6 7 8 9 10