Targeting of protein kinase a to testicular and pancreatic peroxisomes by a novel a-kinase anchoring protein: AKAP220

The specificity of cAMP dependent protein kinase (PKA) for a hormone can be achieved through compartmentalization of the kinase with particular protein substrates. PKA is maintained in these subcellular compartments via an interaction of the regulatory subunit (RII) with an A-kinase anchoring protein (AKAP). Using an interaction cloning strategy with an RII alpha protein probe, we have isolated cDNAs encoding a novel 1129-amino acid protein (AKAP220) which contains an RII binding region and a peroxisome targeting motive. Peptide studies indicate that the RII binding region is located at residues 905-918 which is predicted to form an amphipathic helix. Northern analysis of AKAP220 detected a 9-8 and 7.3 kB message in several rat tissues, however, the message was predomiately expressed in the testis. In addition, Western analysis of rat tissues show the protein to be expressed in the testis and in a pancreatic beta cell line, RIN m5F and runs on SDS-PAGE at 220kDa. The complex of RII and AKAP220 was identified in rat testis by immunoprecipitating with anti-AKAP220 antibodies and showing the presence of PKA by Western blot, RII overlay and PKA activity assays. The specific activity of PKA increased from 3.8±1.7 pmol / mg / min in the total lysate to 3.0±1.2 nmol/ mg/ min in the immunoprecipitate. AKAP 220 and RII were co-localized in the testicular cell lines, TM3 and TM4, and RIN mSF cells as detected by immunohistochemistry and confocal microscopy. The subcellular distribution of the complex corresponded with peroxisome distribution. This association was confirmed by confocal microscopy on TM 4 cells after labeling with anti-AKAP220 and anti-peroxisomal protein (70 kDa). Collectively, these results suggest that the novel protein, AKAP220, associates with tbe regulatory subunit of PKA and targets it to peroxisomes in rat testis and pancreatic beta cells. The functional role of peroxisome-targeted PKA is being evaluated.