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

Darren D. Browning, Marisa Mc Shane, Caroline Marty, Richard D. Ye

Research output: Contribution to journalArticle

42 Scopus citations

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 PKG1α (G1αR-GFP) or the catalytic domains of PKG1α (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 PKG1α-GFP fusion proteins were found to have constitutive activity both in vivo and in vitro. The G1αR-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, G1αR-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 G1αR-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 G1αR-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, PKG1α 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.

Original languageEnglish (US)
Pages (from-to)13039-13048
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number16
DOIs
Publication statusPublished - Apr 20 2001

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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