High-affinity binding and localization of the cyclic GMP-dependent protein kinase with the intermediate filament protein vimentin.
The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform (subunit M(r) congruent to 78,000) of this enzyme mediates the effects of cGMP to relax contracted vascular smooth muscle preparations. In this study, we have addressed the hypothesis that the cGMP kinase is anchored to intracellular proteins which might serve to target cGMP kinase to protein substrates. Using a gel overlay technique, immunoprecipitation, and a fluorescence binding assay for cGMP kinase, we have identified vimentin as a high-affinity and specific binding protein for cGMP kinase. Binding of cGMP kinase to vimentin is reversible and stoichiometric (one cGMP kinase dimer/vimentin dimer) with a KD of approximately 49 nM. The site of high-affinity binding between cGMP kinase and vimentin did not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase did not compete for high-affinity binding. Neither the proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment bound vimentin with high affinity, suggesting that the cGMP kinase dimer was necessary for the interaction. Vimentin was readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment even though the monomeric 69-kDa fragment was catalytically active toward other substrates such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
MacMillan-Crow
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LA
Department of Pharmacology and Pathology, University of Alabama at Birmingham, 35294-0019.
Lincoln
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TM
D000818:Animals; D002417:Cattle; D002451:Cell Compartmentation; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D007150:Immunohistochemistry; D008954:Models, Biological; D008956:Models, Chemical; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014746:Vimentin / Q000378:metabolism*
D010446:Peptide Fragments; D014746:Vimentin; D017869:Cyclic GMP-Dependent Protein Kinases; D002918:Chymotrypsin