Functional analysis of type 1α cGMP-dependent protein kinase using green fluorescent fusion proteins

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 alpha (G1 alphaR-GFP) or the catalytic domains of PKG1 alpha: (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 alpha -GFP fusion proteins were found to have constitutive activity both in vivo and in vitro. The G1 alphaR-GFP protein was found to dimerize with endogenous type I 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 alphaR-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 PKC-GFP protein was present in a more centralized location, whereas the G1LYR-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 alphaR-GFP and the PKG1 alpha -GFP fusion proteins colocalized with vasodilator-stimulated phosphoprotein (VASP), These constructs thus represent novel tools with which to visualize inactive, and activated, PKG1 alpha 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 PKC is found in dynamic membrane regions in association with VASP.