scholarly journals Increasing cGMP-dependent protein kinase activity attenuates unilateral ureteral obstruction-induced renal fibrosis

2014 ◽  
Vol 306 (9) ◽  
pp. F996-F1007 ◽  
Author(s):  
Wenpeng Cui ◽  
Hasiyeti Maimaitiyiming ◽  
Xinyu Qi ◽  
Heather Norman ◽  
Qi Zhou ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Renal Fibrosis ◽  
Cell Function ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Tubular Cells ◽  
Cgmp Dependent Protein Kinase ◽  
Proximal Tubular ◽  
Dependent Protein

Our previous studies support the protective effect of cGMP and cGMP-dependent protein kinase I (PKG-I) pathway on the development of renal fibrosis. Therefore, in the present studies, we determined whether pharmacologically or genetically increased PKG activity attenuates renal fibrosis in a unilateral ureteral obstruction (UUO) model and also examined the mechanisms involved. To increase PKG activity, we used the phosphodiesterase 5 inhibitor sildenafil and PKG transgenic mice. UUO model was induced in wild-type or PKG-I transgenic mice by ligating the left lateral ureteral and the renal fibrosis was observed after 14 days of ligation. Sildenafil was administered into wild-type UUO mice for 14 days. In vitro, macrophage and proximal tubular cell function was also analyzed. We found that sildenafil treatment or PKG transgenic mice had significantly reduced UUO-induced renal fibrosis, which was associated with reduced TGF-β signaling and reduced macrophage infiltration into kidney interstitial. In vitro data further demonstrated that both macrophages and proximal tubular cells were important sources of UUO-induced renal TGF-β levels. The interaction between macrophages and tubular cells contributes to TGF-β-induced renal fibrosis. Taken together, these data suggest that increasing PKG activity ameliorates renal fibrosis in part through regulation of macrophage and tubular cell function, leading to reduced TGF-β-induced fibrosis.

scholarly journals Increasing cGMP-dependent protein kinase I activity attenuates cisplatin-induced kidney injury through protection of mitochondria function

2013 ◽  
Vol 305 (6) ◽  
pp. F881-F890 ◽  
Author(s):  
Hasiyeti Maimaitiyiming ◽  
Yanzhang Li ◽  
Wenpeng Cui ◽  
Xiaopeng Tong ◽  
Heather Norman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tubular Damage ◽  
Dependent Protein Kinase ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Renal Tubular

Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear, and the renoprotective agents during cisplatin treatment are still lacking. Here, we demonstrated that the expression and activity of cGMP-dependent protein kinase-I (PKG-I) were reduced in cisplatin-treated renal tubular cells in vitro as well as in the kidney tissues from cisplatin-treated mice in vivo. Increasing PKG activity by both pharmacological and genetic approaches attenuated cisplatin-induced kidney cell apoptosis in vitro. This was accompanied by decreased Bax/Bcl2 ratio, caspase 3 activity, and cytochrome c release. Cisplatin-induced mitochondria membrane potential loss in the tubular cells was also prevented by increased PKG activity. All of these data suggest a protective effect of PKG on mitochondria function in renal tubular cells. Importantly, increasing PKG activity pharmacologically or genetically diminished cisplatin-induced tubular damage and preserved renal function during cisplatin treatment in vivo. Mitochondria structural and functional damage in the kidney from cisplatin-treated mice was inhibited by increased PKG activity. In addition, increasing PKG activity enhanced ciaplatin-induced cell death in several cancer cell lines. Taken together, these results suggest that increasing PKG activity may be a novel option for renoprotection during cisplatin-based chemotherapy.

Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro

2003 ◽  
Vol 284 (4) ◽  
pp. H1388-H1397 ◽  
Author(s):  
Hyun Kook ◽  
Hiroshi Itoh ◽  
Bong Seok Choi ◽  
Naoki Sawada ◽  
Kentaro Doi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Physiological Concentration ◽  
Cell Numbers ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Regeneration In Vitro

Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiological concentration (10−11mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations.

scholarly journals Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Il Young Kim ◽  
Min Young Lee ◽  
Mi Wha Park ◽  
Eun Young Seong ◽  
Dong Won Lee ◽  
...  
Keyword(s):  
Murine Model ◽  
Ureteral Obstruction ◽  
Renal Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Type I ◽  
Wild Type ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Hk2 Cells

We investigated the role of Akt1, one of the three isoforms of Akt, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). We subjected wild type and Akt1−/− mice to UUO. The Akt1 gene was silenced in vitro using short hairpin RNA delivered via a lentiviral vector in human proximal tubular cells (HK2 cells) and kidney fibroblasts (NRK-49F cells). The obstructive kidneys of Akt1−/− mice showed more severe tubulointerstitial fibrosis than those of wild type mice. The expression of fibronectin and type I collagen was significantly increased in obstructed kidneys of Akt1−/− mice compared to those of wild type mice. The important finding was that the expression of transforming growth factor β1 (TGFβ1) was significantly increased in the Akt1−/− mice compared to the wild type mice. The knockdown of Akt1 enhanced the expression of TGFβ1 in HK2 cells. Interestingly, the upregulation of TGFβ1 due to genetic knockdown of Akt1 was associated with activation of signal transducer and activator of transcript 3 (STAT3) independently of the Smad pathway in NRK-49F and HK2 cells. Immunohistochemical staining also showed that expression of phosphorylated STAT3 was more increased in Akt1−/− mice than in wild type mice after UUO. Additionally, the deletion of Akt1 led to apoptosis of the renal tubular cells in both in vivo and in vitro studies. Conclusively, these results suggest that the deletion of Akt1 may contribute to renal fibrosis via induction of the TGFβ1/STAT3 pathway in a murine model of UUO.

scholarly journals The Flagellar Motility ofChlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions

2006 ◽  
Vol 17 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Chun Yang ◽  
Pinfen Yang
Keyword(s):  
Regulatory Proteins ◽  
Flagellar Motility ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Wide Range ◽  
Anchoring Protein ◽  
Radial Spokes ◽  
Dependent Protein ◽  
Cilia And Flagella

Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.

scholarly journals Depletion of cyclic‐GMP levels and inhibition of cGMP‐dependent protein kinase activate p21 Cip1 /p27 Kip1 pathways and lead to renal fibrosis and dysfunction

2020 ◽  
Vol 34 (9) ◽  
pp. 11925-11943
Author(s):  
Subhankar Das ◽  
Kandasamy Neelamegam ◽  
Whitney N. Peters ◽  
Ramu Periyasamy ◽  
Kailash N. Pandey
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Renal Fibrosis ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
P27 Kip1

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity

1985 ◽  
Vol 5 (7) ◽  
pp. 1772-1779
Author(s):  
M A Snyder ◽  
J M Bishop ◽  
J P McGrath ◽  
A D Levinson
Keyword(s):  
Binding Site ◽  
Kinase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Specific Immunoglobulin ◽  
Dependent Protein ◽  
Infected Cells

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.

The Role of the cGMP-Dependent Protein Kinase II in Platelet Activation and In Vivo Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 653-653
Author(s):  
Zhenyu Li ◽  
Guoying Zhang ◽  
Hong Yin ◽  
Robert Feil ◽  
Franz Hofmann ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Low Dose ◽  
Bleeding Time ◽  
Wild Type ◽  
Rt Pcr ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Abstract Although it was previously believed that the intracellular secondary messenger cGMP inhibits platelets, we have recently shown that cGMP-dependent protein kinase I (PKG I) in fact plays a stimulatory role in platelet activation. However, there are apparent differences between the PKG inhibitors and PKG I knockout in their effects on platelet activation. PKG inhibitors are more potent in inhibiting platelet activation than PKG I knockout. More importantly, although platelet secretion and aggregation induced by collagen were inhibited by PKG inhibitors, they are not significantly affected in PKG I knockout platelets. There are two types of PKG, PKG I and PKG II. PKG II has not been previously described in platelets. Here we show that PKG II mRNA is expressed in platelets using RT-PCR with primers specific for a C-terminal fragment of human PKG II cDNA. We further cloned the complete cDNA of human PKG II by RT-PCR using the purified human platelet mRNA as a template. Furthermore, PKG II from platelet lysates was pulled down by cGMP conjugated agarose beads and detected by western blot using a polyclonal antibody against PKG II. These data indicate that PKG II is expressed in platelets. To investigate the role of PKG II in platelet activation, washed wild type or PKG II knockout (PKG II−/−) mouse platelets in tyrode’s solution were exposed to platelet agonists. Platelet aggregation and ATP secretion induced by low concentrations of collagen were significantly reduced in PKG II deficient mice, indicating that PKG II plays important roles in collagen-induced platelet activation. PKG II−/− platelets also showed reduced aggregation and secretion to low dose of a thromboxane A2 (TXA2) analog, U46619. However, low dose thrombin-induced platelet activation was not negatively affected in PKG II−/− platelets, but was inhibited in PKG I−/− platelets. To evaluate the in vivo role of PKG II, we compared in vivo thrombus formation of wild type and PKG II knockout mice using the FeCl3-injured carotid artery thrombosis model. The time to the formation of stable thrombus in PKG II−/− mice (median, 420.0 seconds, n=15) is significantly prolonged compared to wild type mice (median, 321.0 seconds, n=15) (p=0.031). Tail-bleed time analysis also indicated a remarkably prolonged bleeding time in PKG II−/− mice (the median bleeding time was 73.50 seconds (n=18) in wild type mice, 454.50 seconds (n=20) in PKG II knockout mice) (p=0.0008). Thus, PKG II plays an important role in promoting platelet activation, thrombosis and hemostasis. PKG I and PKG II have differential roles in platelet activation induced by different platelet agonists.

In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium

1993 ◽  
Vol 106 (4) ◽  
pp. 1369-1376 ◽  
Author(s):  
C.E. Walczak ◽  
D.L. Nelson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Sucrose Gradient ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Ciliary Protein ◽  
Dependent Protein ◽  
Camp And Cgmp

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.

scholarly journals Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase

2018 ◽  
Vol 293 (43) ◽  
pp. 16791-16802 ◽  
Author(s):  
Jessica L. Sheehe ◽  
Adrian D. Bonev ◽  
Anna M. Schmoker ◽  
Bryan A. Ballif ◽  
Mark T. Nelson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Specific Substrate ◽  
Type I ◽  
Amino Acid Residues ◽  
Dependent Protein Kinase ◽  
Constitutive Activation ◽  
Channel Characteristics ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2. In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα–specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

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