Abstract
Classical A kinase anchor proteins (AKAPs) preferentially tether type II protein kinase A (PKAII) isoforms to sites in the cytoskeleton and organelles. It is not known if distinct proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In Caenorhabditis elegans, a single type I PKA mediates all aspects of cAMP signaling. We have discovered a cDNA that encodes a binding protein (AKAPCE) for the regulatory subunit (RCE) of C. elegans PKAICE. AKAPCE is a novel, highly acidic RING finger protein composed of 1,280 amino acids. It binds RI-like RCE with high affinity and neither RIIalpha nor RIIbeta competitively inhibits formation of AKAPCE.RCE complexes. The RCE-binding site was mapped to a segment of 20 amino acids in an N-terminal region of AKAPCE. Several hydrophobic residues in the binding site align with essential Leu and Ile residues in the RII-selective tethering domain of prototypic mammalian AKAPs. However, the RCE-binding region in AKAPCE diverges sharply from consensus RII-binding sites by inclusion of three aromatic amino acids, exclusion of a highly conserved Leu or Ile at position 8 and replacement of C-terminal hydrophobic amino acids with basic residues. AKAPCE.RCE complexes accumulate in intact cells.
Highlights
A kinase anchor proteins (AKAPs)1 avidly bind regulatory subunits (RII and RII␣) of the type II isoforms of cAMP-dependent protein kinase (PKAII and PKAII␣) [1,2,3]
RI-specific anchor proteins? If so, what is the biochemical basis for the binding and anchoring of RI (PKAI)? To what degree are structures of RI anchor proteins homologous with or divergent from RII-selective AKAPs? The nematode Caenorhabditis elegans provides an advantageous system for addressing these questions
Transfection of AV-12 Cells—Full-length RCE and AKAPCE cDNA inserts were excised from recombinant, pBluescript plasmids by digestion with NotI and ApaI and ligated into the pCIS2 mammalian expression vector [30], which was cleaved with the same enzymes
Summary
Isolation of cDNAs Encoding AKAPCE—Approximately 500,000 plaques from a C. elegans cDNA library in the expression vector ZAPII (CLONTECH) were screened by the procedure of Bregman et al [18, 19]. Transfection of AV-12 Cells—Full-length RCE and AKAPCE cDNA inserts were excised from recombinant, pBluescript plasmids by digestion with NotI and ApaI and ligated into the pCIS2 mammalian expression vector [30], which was cleaved with the same enzymes. This placed the cDNAs downstream from a powerful cytomegalovirus promoter and upstream from a poly(A) addition signal. Expression and Purification of a GST Fusion Protein That Contains the RCE-binding Domain—A fragment of AKAPCE cDNA (nucleotides 526 –1227, Fig. 2A) that encodes amino acids 176 – 409 in the anchor protein was cloned into the expression plasmid pGEX-KG. The Western blots were probed with anti-AKAPCE IgGs and AKAPCE1⁄7IgG complexes were visualized as described above
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call