Functional analysis of type 1alpha 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 PKG1alpha (G1alphaR-GFP) or the catalytic domains of PKG1alpha (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 PKG1alpha-GFP fusion proteins were found to have constitutive activity both in vivo and in vitro. The G1alphaR-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, G1alphaR-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 G1alphaR-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 G1alphaR-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, PKG1alpha 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.
Browning
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Department of Pharmacology (MC 868), University of Illinois at Chicago, Chicago, Illinois 60612, USA. dbrowning@mail.mcg.edu
Mc Shane
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Marty
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D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020545:Respiratory Mucosa; D014162:Transfection
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017869:Cyclic GMP-Dependent Protein Kinases