Abstract
Serum amyloid A (SAA) activating factor-1 (SAF-1) is an inducible transcription factor that plays a key role in the regulation of several inflammation-responsive genes including SAA and matrix metalloproteinase-1. Increased synthesis of SAA and matrix metalloproteinase-1 is associated with pathogenesis of several diseases including amyloidosis, arthritis, and atherosclerosis. Previously, we showed in vivo interaction of SAF-1 and protein kinase A (PKA) and presented evidence for induction of SAF-1-regulated genes by a PKA signaling pathway. Here we demonstrate a mechanism by which PKA increases functional activities of SAF-1. Site-directed mutagenesis and phosphorylation analyses revealed two sites in the SAF-1 protein, serine 187 and threonine 386, as the target of PKA. Interestingly, mutation of both PKA phosphorylation sites created a highly active SAF-1 protein with high DNA-binding ability. Furthermore, we found that terminal deletion of SAF-1 protein from either end creates SAF-1 isoforms that are highly transcriptionally active. Partial proteolysis experiments indicated that unphosphorylated and phosphorylated SAF-1 proteins are structurally distinct. Together these results suggest that under native condition, N and C termini of SAF-1 are engaged in an inhibitory intramolecular interaction. PKA-mediated phosphorylation increases transcriptional activity of SAF-1 by unmasking the DNA-binding domain.
Highlights
As protein kinase A (PKA) is one of the key enzymes involved in the regulation of many biological processes it is reasonable to predict that PKA may be a key effector in modulating expression of genes regulated by serum amyloid A activating factor (SAF)-1
Because the reporter used in these assays was constructed using tandem multiple copies of short SAF-1 DNA-binding elements, we further verified the inducive effects of PKA by using a natural promoter, 0.6 Serum amyloid A (SAA)-CAT3 reporter gene that contained a 600-bp upstream promoter region of rabbit SAA gene [2, 23]
Previous investigations suggested that interaction between SAF-1 and PKA-C␣ may regulate the functional activity of SAF-1 [16, 19]
Summary
Tion [1, 2]. Persistent high levels of SAA are linked to various pathophysiological conditions, including amyloidosis, rheumatoid arthritis, and atherosclerosis [3, 4]. Bacterially expressed wild-type and mutant SAF-1 proteins were phosphorylated with 1.0 unit of the catalytic subunit of PKA (Calbiochem-Novabiochem) in a volume of 10 l prior to the DNA-binding assay. The transcription of GAL4-CAT reporter was further enhanced when cells were co-transfected with PKA expression plasmid DNA indicating that PKA directly increases transactivating potential of SAF-1.
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