AbstractsBiology & Animal Science

Abstract

Summary Reversible protein phosphorylation is one of the most important mechanisms for regulation and coordination of cellular processes, and is uttered through the actions of protein kinases and phosphatases. One of the best characterised protein kinases is cyclic AMP-dependent protein kinase A (PKA). PKA is a holoenzyme which consists of a regulatory (R) dimer and two catalytic (C) subunits. Humans have at least four genes that express four isoforms of the C subunits, designated Cá, Câ, Cã and PrKX. Both the Cá and the Câ genes express splice variants, all of which differ in the N-terminal part encoded by the exons located upstream of exon 2. How specificity is maintained in the cAMP-PKA pathway is not fully understood but involves targeting of the R subunit dimer to subcellular structures through proteins that are termed A kinase anchoring proteins (AKAPs). There is one problem with maintaining specificity by using this model. That is, when the PKA holoenzyme is challenged with cAMP the C subunits are released from the R-AKAP complex, which will leave the C subunits free to unspecifically phosphorylate targets in the cytosol and the nucleus. However, this is not the case, and hence it has been speculated as to the existence of C subunit binding proteins both in the cytosol and nucleus. The C binding proteins will target the C subunit in close proximity to its substrate, and in this way increase the level of specificity in the cAMP-PKA signalling pathway. Indeed, today seven C binding proteins have been identified, of which three are cytosolic; inhibitor of NF-êB (IêB), Caveolin-1, and p75 neurotrophin receptor (p75NTR), two are strictly nuclear; homologous to AKAP95 (HA95) and A kinase interacting protein (AKIP) and last is the protein kinase inhibitor (PKI) which is located both to the cytosol and the nucleus. In order to identify more C binding partners, Câ2 was used as bait in a yeast two-hybrid screen of a human peripheral blood cell cDNA library. This was performed by the company Dualsystems Biotech. In addition to the PKA R subunit RI, a novel potential binding partner was identified as the nearly ubiquitously expressed eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). eEF1A1 is associated with ribosome activity and protein synthesis and brings the incoming aminoacyl-tRNA in a GTP-dependent manner to a site within the ribosome referred to as the aminoacyl (A) site. In this Master thesis the eEF1A1 was further characterised by applying PCR cloning, eukaryote expression, immunofluoresence and immunoprecipitation. This demonstrated that eEF1A1 was mainly located in the cell cytosol, and associated with all the various PKA C subunits tested which included Câ1, Câ2, Câ4 as well as Cá1. Our data implies that we have identified a novel binding partner for the C subunit of PKA expressed in all tissues except differentiated skeletal muscle, linking the regulatory role of the cAMP-PKA signalling pathway to protein synthesis.