Functional Analysis of Type 1α cGMP-dependent Protein Kinase Using Green Fluorescent Fusion Proteins

Abstract

The cGMP-dependent protein kinases (PKGs) are ubiquitous effector enzymes that regulate a variety of physiological processes in response to nitric oxide and natriuretic agonists. We have constructed green fluorescent fusion proteins (GFP) using full-length (PKG-GFP) and truncations encoding either the regulatory domain of PKG1␣ (G1␣R-GFP) or the catalytic domains of PKG1␣ (GFP-G1C) to examine the enzymatic properties and intracellular location. When transiently transfected into mammalian cells, these constructs were detected on Western blots at the expected sizes using anti-GFP antibodies. The GFP-G1C and the full-length PKG1␣-GFP fusion proteins were found to have constitutive activity both in vivo and in vitro. The G1␣R-GFP protein was found to dimerize with endogenous type 1 PKG and behaved in a dominant negative manner both in vivo and in vitro. When expressed transiently in either HEK-293 or A549 epithelial cells, the fusion proteins encoding the amino-terminal regulatory domains (PKG-GFP, G1␣R-GFP) were present in the cytosol and were rarely observed in the nucleus. In contrast, the GFP-G1C (lacking regulatory domains) concentrated in the nucleus. Of the fusion proteins containing the regulatory region, the constitutive PKG-GFP protein was present in a more centralized location, whereas the G1␣R-GFP protein colocalized with F-actin on stress fibers and in dynamic regions of the plasma membrane. Microscopic and immunoprecipitation studies indicated that both the G1␣R-GFP and the PKG-GFP fusion proteins colocalized with vasodilator-stimulated phosphoprotein (VASP). These constructs thus represent novel tools with which to visualize inactive, and activated, PKG1␣ in vivo, and we have used them to demonstrate two functionally independent domains. In addition, we show for the first time in living cells that PKG is found in dynamic membrane regions in association with VASP.

Regulation of Gene Expression by Cyclic GMP

“…95,99 -102 However, other investigators found no evidence of PKG nuclear translocation in primary VSMCs, HEK293, and CV-1 cells, or observed nuclear PKG only in a minority of the cell population. 97,103,104 Based on recent findings, we have speculated that PKG may be retained in extranuclear compartments by binding to cell…”

Nitric Oxide Decreases the Expression of Endothelin-Converting Enzyme-1 Through mRNA Destabilization

“…28,29 Additionally, the role of the 3Ј-UTR region was proven by using ECE-1 transgenes with (pCMV6-UTR-ECE1) or without this region (pCMV6-ECE1) (Please see Supplemental data). PKG involvement was studied using a dominant-negative form and a constitutively active construct of PKG-1␣, 30,31 which were kindly donated by Dr D. Browning (Medical College of Georgia, Augusta, GA).…”

Activation of the Mitogen Activated Protein Kinase Extracellular Signal-Regulated Kinase 1 and 2 by the Nitric Oxide–cGMP–cGMP-Dependent Protein Kinase Axis Regulates the Expression of Matrix Metalloproteinase 13 in Vascular Endothelial Cells

“…7A, bottom). Although a nonspecific interaction between PKG and Raf-1 cannot be definitively excluded, the capacity of the catalytic region of PKG1␣ to interact with the full-length and the regulatory regions has been previously shown (Browning et al, 2001). In addition, the complex is inducible and time-sensitive, supporting the idea that Raf-1 is a target of PKG in the aortic endothelium (Fig.…”