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1.
To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.  相似文献   

2.
Phosphorylation of the cystic fibrosis transmembrane conductance regulator.   总被引:17,自引:0,他引:17  
Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.  相似文献   

3.
We have examined cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein (Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. (1991) Biochem. Biophys. Res. Commun. 175, 192-198). Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labeled both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites (peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogues and specific inhibitors of PKG and PKA induced in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylated Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo.  相似文献   

4.
Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.  相似文献   

5.
Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the beta 2 subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologously expressed rat beta 2a subunit was phosphorylated at two sites in vitro by purified PKA. This beta 2a subunit contains two potential consensus sites for PKA-mediated phosphorylation at Thr164 and Ser591. However, upon mutation of both of these residues to alanines, the beta 2a subunit remained a good substrate for PKA. The actual sites of phosphorylation on the beta 2a subunit were identified by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a bacterially expressed beta 2a subunit demonstrated that this subunit was phosphorylated similarly to the beta 2 subunit isolated from heart tissue and that the phosphorylation sites were contained in the unique C-terminal region. Microsequencing identified three serine residues, each of which conformed to loose consensus sites for PKA-mediated phosphorylation. Mutation of these residues to alanines resulted in the loss of the PKA-mediated phosphorylation of the beta 2a subunit. The results suggest that phosphorylation of the beta 2a subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus and not at either of the two strong consensus sites for PKA. The results also highlight the danger of assuming that consensus sites represent actual sites of phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved in most beta 2 subunit isoforms and thus represent potential sites for regulation of channel activity. The sites phosphorylated by PKA are not substrates for protein kinase C (PKC), as the mutated beta 2 subunits lacking PKA sites remained good substrates for PKC.  相似文献   

6.
It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.  相似文献   

7.
Huang SY  Tsai ML  Wu CJ  Hsu JL  Ho SH  Chen SH 《Proteomics》2006,6(6):1722-1734
Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states.  相似文献   

8.
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.  相似文献   

9.
Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.  相似文献   

10.
Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.  相似文献   

11.
This study examined the expression of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP(3)Rs and inhibit IP(3)-dependent Ca(2+) release, which mediates the initial phase of agonist-induced contraction. PKG-Ialpha and PKG-Ibeta were expressed in gastric smooth muscle cells, together with IP(3)-R-associated cG-kinase substrate, a protein that couples PKG-Ibeta to IP(3)R-I. IP(3)R-I and IP(3)R-III were also expressed, but only IP(3)R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Ialpha in vitro showed that PKA phosphorylated the same site as PKG (presumably S(1755)) and an additional PKA-specific site (S(1589)). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP(3)R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP(3)R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP(3)R-I in intact muscle cells. The pattern of IP(3)R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Ialpha. Phosphorylation of IP(3)R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP(3)-induced Ca(2+) release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP(3)R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP(3)-dependent Ca(2+) release.  相似文献   

12.
The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K(m) and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.  相似文献   

13.
Activation of NMDA receptors leads to activation of cAMP-dependent protein kinase (PKA). The main substrates phosphorylated by PKA following NMDA receptor activation remain unidentified. The aim of this work was to identify a major substrate phosphorylated by PKA following NMDA receptor activation in cerebellar neurones in culture, and to assess whether this phosphorylation may be involved in neuronal death induced by excessive NMDA receptor activation. The main PKA substrate following NMDA receptor activation was identified by MALDI-TOFF fingerprinting as the nuclear protein, matrin 3. PKA-mediated phosphorylation of matrin 3 is followed by its degradation. NMDA receptor activation in rat brain in vivo by ammonia injection also induced PKA-mediated matrin 3 phosphorylation and degradation in brain cell nuclei. Blocking NMDA receptors in brain in vivo with MK-801 reduced basal phosphorylation of matrin 3, suggesting that it is modulated by NMDA receptors. Inhibition of PKA with H-89 prevents NMDA-induced phosphorylation and degradation of matrin 3 as well as neuronal death. These results suggest that PKA-mediated phosphorylation of matrin 3 may serve as a rapid way of transferring information from synapses containing NMDA receptors to neuronal nuclei under physiological conditions, and may contribute to neuronal death under pathological conditions.  相似文献   

14.
Careful regulation of the Wnt-Beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway.  相似文献   

15.
Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).  相似文献   

16.
The intracellular domain of the C type natriuretic peptide receptor (NPRC) contains one threonine and several serine residues where phosphorylation is thought to occur. Several phosphorylation consensus sequences for various kinases have been identified within the intracellular domain of NPRC, but the exact residues that are phosphorylated and the specific kinases responsible for their phosphorylation have not been thoroughly defined. Here we introduce a recombinant GST fusion protein and a rat gastric mucosa (RGM1) cell line as molecular tools to study the phosphorylation state of NPRC in vitro and in vivo, respectively. We utilize a previously characterized polyclonal antibody against NPRC to probe for total NPRC protein and various phosphospecific and substrate motif antibodies to probe for phosphorylation of NPRC. Phosphoprotein staining reagents were used with a phosphoprotein control set to detect phosphorylation of NPRC at serine and threonine residues. Recombinant GST‐NPRC fusion protein was phosphorylated in vitro by RGM1 lysate in the presence of adenosine‐5'‐triphosphate (ATP). Western blot analysis using a monoclonal phospho‐Thr antibody, which exclusively detects phosphorylated threonine residues, and does not cross‐react with phosphorylated serine residues revealed NPRC immunoprecipitated from RGM1 lysate is phosphorylated on a threonine residue. Global analysis of the entire rat NPRC sequence using a protein kinase A (PKA) prediction algorithm, identified five putative PKA phosphorylation sites containing a serine residue and one containing a threonine residue, Thr 505. Taken together, the data presented here suggest that rat NPRC is a substrate for PKA and Thr 505 located within the intracellular domain of NPRC is a likely candidate site for the phosphorylation. J. Cell. Biochem. 110: 985–994, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

17.
Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.  相似文献   

18.
Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C   总被引:1,自引:0,他引:1  
Chitaley K  Chen L  Galler A  Walter U  Daum G  Clowes AW 《FEBS letters》2004,556(1-3):211-215
Vasodilator-stimulated phosphoprotein (VASP), an actin binding protein localized to areas of focal contacts, is a substrate for the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP)-dependent protein kinases (PKA, PKG). In this study, we show that serum stimulation of vascular smooth muscle cells (SMCs) induces VASP phosphorylation on Ser157, in a mechanism not dependent on PKA or PKG. We tested the possibility that protein kinase C (PKC), a regulator of cytoskeletal function, is involved. PKC inhibition or down-regulation prevented serum-induced phosphorylation of VASP at Ser157 in rat vascular SMCs. Additionally, recombinant PKCalpha directly phosphorylated Ser157 on VASP. In summary, our data support the hypothesis that PKC phosphorylates VASP and mediates serum-induced VASP regulation.  相似文献   

19.
The role of protein kinase, in particular cyclic GMP-dependent protein kinase (PKG), in the control of chemotaxis was studied in Tetrahymena thermophila using the membrane-permeable cGMP analogue 8-bromo-cGMP and the NO-generator sodium nitroprusside (SNP) that stimulates cGMP production by activating guanylate cyclase. Stimulation of chemoattraction was observed in the presence of 8-bromo-cGMP and nitroprusside when used in 10–100 μM concentrations in vivo. In vitro stimulation of ciliary membrane PKG activity was observed when using similar concentrations of cGMP or 8-bromo-cGMP to those in the in vivo experiments. In contrast, the protein kinase flavonol inhibitors quercitin and kaempherol block chemoattraction and reduce ciliary membrane PGK activity in vitro. For the inhibition of PKG, the IC-50 s for quercitin and kaempherol are 22 and 19 μM, respectively. The results suggest a modulating function of PKG on adaptory processes in cilia-mediated chemotaxis.

The ciliary membrane-associated PKG was partially characterized. Without added external protein kinase substrate in vitro, an endogenous ciliary membrane kinase activity showed phosphorylation of 55 and 97 kDa Triton-X-100 soluble proteins when analyzed by SDS-PAGE under reducing conditions and with 32P-γ-ATP as phosphorylation donor. Phosphoamino acid analysis of PKG-phosphorylated proteins showed 32P-phosphate labeling of serine and threonine residues. Ciliary membrane-associated PKG was further purified by carboxy-methyl-sephadex-column chromatography. The membrane enzyme was Mg2++-dependent and had a pH optimum at 6.4. The carboxy-methyl-sephadex-eluted PKG was analyzed by electrophoresis on sodium dodecyl sulphate polyacrylamide gels showing a molecular weight of 70–75 kDa.  相似文献   


20.
Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein kinase A (PKA) phosphorylation sites. The neural network was trained with a positive set of 258 experimentally verified PKA phosphorylation sites. The predictions by NetPhosK were validated using four novel PKA substrates: Necdin, RFX5, En-2, and Wee 1. The four proteins were phosphorylated by PKA in vitro and 13 PKA phosphorylation sites were identified by mass spectrometry. NetPhosK was 100% sensitive and 41% specific in predicting PKA sites in the four proteins. These results demonstrate the potential of using integrated computational and experimental methods for detailed investigations of the phosphoproteome.  相似文献   

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