Protein kinase A signaling pathway regulates transcriptional activity of SAF-1 by unmasking its DNA-binding domains.
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.
Ray
Alpana
A
Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri 65211, USA. rayal@missouri.edu
Ray
Papiya
P
Guthrie
Nicole
N
Shakya
Arvind
A
Kumar
Deepak
D
Ray
Bimal K
BK
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017930:Genes, Reporter; D010766:Phosphorylation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology*; D013583:Synovial Membrane / Q000166:cytology / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C076035:c-MYC-associated zinc finger protein; D015500:Chloramphenicol O-Acetyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases