scholarly journals A substitution in cGMP-dependent protein kinase 1 associated with aortic disease induces an active conformation in the absence of cGMP

2020 ◽  
Vol 295 (30) ◽  
pp. 10394-10405
Author(s):  
Matthew H. Chan ◽  
Sahar Aminzai ◽  
Tingfei Hu ◽  
Amatya Taran ◽  
Sheng Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Leucine Zipper ◽  
Aortic Aneurysms ◽  
Cell Phenotype ◽  
Active Conformation ◽  
Dependent Protein Kinase ◽  
Basal Activity ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

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 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.

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.

MYPT1 mutants demonstrate the importance of aa 888–928 for the interaction with PKGIα

2007 ◽  
Vol 292 (1) ◽  
pp. C432-C439 ◽  
Author(s):  
Allison M. Given ◽  
Ozgur Ogut ◽  
Frank V. Brozovich
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Myosin Light Chain ◽  
Leucine Zipper ◽  
Dependent Protein Kinase ◽  
Smooth Muscle Tissue ◽  
Nitric Oxide Signaling ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

During nitric oxide signaling, type Iα cGMP-dependent protein kinase (PKGIα) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIα interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH2-terminal LZ of PKGIα (HK Surks and ME Mendelsohn. Cell Signal 15: 937–944, 2003; HK Surks et al. Science 286: 1583–1587, 1999), but we previously showed that PKGIα interacts with LZ-positive (LZ+) and LZ-negative (LZ−) MYPT1 isoforms ( 13 ). Interestingly, PKGIα is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772–14777, 2000), and there is an RK motif within the aa 888–928 sequence of MYPT1 in LZ+ and LZ− isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIα interaction, we designed four MYPT1 fragments that contained both the aa 888–928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888–928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888–928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888–928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGIα in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916–917 (R916K917) to AA decreased binding of MYPT1 to PKGIα in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R916K917 to E916E917 eliminated binding, suggesting that one factor important for the PKGIα-MYPT1 interaction is the charge at aa 916–917. These results suggest that, during cGMP-mediated signaling, aa 888–928 of MYPT1 mediate the PKGIα-MYPT1 interaction.

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