The catalytic domain of the cGMP-dependent protein kinase Iα modulates the cGMP-binding characteristics of its regulatory domain

Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

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cGMP-dependent protein kinase protects cGMP from hydrolysis by phosphodiesterase-5

Biochemical Journal ◽

10.1042/bj20030107 ◽

2003 ◽

Vol 372 (2) ◽

pp. 419-426 ◽

Cited By ~ 26

Author(s):

Jun KOTERA ◽

Kennard A. GRIMES ◽

Jackie D. CORBIN ◽

Sharron H. FRANCIS

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Fold Increase ◽

Binding Affinities ◽

Dependent Protein Kinase ◽

Physiological Concentration ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Pig Coronary Artery ◽

Hydrolysis Of

The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Iα or PKG-Iβ on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Iα strongly inhibited cGMP hydrolysis with an IC50 value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously. By contrast, PKG-Iβ, which has lower affinity for cGMP than does PKG-Iα, inhibited cGMP hydrolysis with an IC50 of approx. 1 μM. Inhibition by PKG-Iα was more effective than that by PKG-Iβ, consistent with their relative affinities for cGMP. Autophosphorylation of PKGs increased their cGMP-binding affinities and their inhibitory effects on PDE5 hydrolysis of cGMP. Autophosphorylation of PKG-Iβ increased its inhibitory potency on PDE5 hydrolysis of cGMP by 10-fold compared with a 2-fold increase upon autophosphorylation of PKG-Iα. The results indicate that cGMP bound to allosteric cGMP-binding sites of PKG is protected from hydrolysis by PDE5 and that persistent protection of cGMP by either non-phosphorylated or autophosphorylated PKGs may be a positive-feedback control to sustain cGMP signalling.

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A structural analysis of the regulatory domain from the cGMP-dependent protein kinase Iα

BMC Pharmacology ◽

10.1186/1471-2210-11-s1-p53 ◽

2011 ◽

Vol 11 (S1) ◽

Author(s):

Brent W Osborne ◽

Andrew T Menke ◽

Donald K Blumenthal ◽

Wolfgang R Dostmann

Keyword(s):

Structural Analysis ◽

Protein Kinase ◽

Regulatory Domain ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

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Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905558116 ◽

2019 ◽

Vol 116 (28) ◽

pp. 14164-14173 ◽

Cited By ~ 11

Author(s):

Majida El Bakkouri ◽

Imène Kouidmi ◽

Amy K. Wernimont ◽

Mehrnaz Amani ◽

Ashley Hutchinson ◽

...

Keyword(s):

Protein Kinase ◽

Conformational Changes ◽

Catalytic Domain ◽

Kinase Domain ◽

Complex Life Cycle ◽

Dependent Protein Kinase ◽

Malaria Parasites ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Developmental Switches

The cyclic guanosine-3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG fromPlasmodium falciparumandPlasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keepPlasmodiumPKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.

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Functional cGMP-Dependent Protein Kinase Is Phosphorylated in Its Catalytic Domain at Threonine-516

Biochemistry ◽

10.1021/bi00040a029 ◽

1995 ◽

Vol 34 (40) ◽

pp. 13152-13158 ◽

Cited By ~ 23

Author(s):

Robert Feil ◽

Josef Kellermann ◽

Franz Hofmann

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

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cDNA Cloning and Gene Expression of Human Type Iα cGMP-Dependent Protein Kinase

Hypertension ◽

10.1161/01.hyp.27.3.552 ◽

1996 ◽

Vol 27 (3) ◽

pp. 552-557 ◽

Cited By ~ 56

Author(s):

Naohisa Tamura ◽

Hiroshi Itoh ◽

Yoshihiro Ogawa ◽

Osamu Nakagawa ◽

Masaki Harada ◽

...

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Cdna Cloning ◽

Dependent Protein Kinase ◽

Human Type ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

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The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia

International Journal of Molecular Sciences ◽

10.3390/ijms22010052 ◽

2020 ◽

Vol 22 (1) ◽

pp. 52

Author(s):

Mirja Koch ◽

Constanze Scheel ◽

Hongwei Ma ◽

Fan Yang ◽

Michael Stadlmeier ◽

...

Keyword(s):

Protein Kinase ◽

Mouse Model ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Cone Photoreceptor ◽

Photoreceptor Degeneration ◽

Dependent Protein Kinase ◽

Genetic Ablation ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

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