Identification and characterisation of novel protein kinase A Cb splice variants expressed in NT2-N cells differentiated by retinoic acid

Abstract

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Humans contain 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 exon 1. It has previously been shown that retinoic acid (RA), which promotes differentiation of the human neuronal precursor cell line Ntera 2 (NT2) to form NT2-N nerve cells, also promotes the induction of a number of nerve cell specific Câ splice variants which may be important for PKA-dependent regulation of nerve cell differentiation and function. In this thesis, we show that RA also induces 6 novel mRNA species from the Câ gene, which lack the nucleotides transcribed from the exon 4. Identification of Câ forms lacking the exon 4 sequence was also confirmed in human brain mRNA. This was concluded using a method that was developed in order to specifically detect low levels of mRNA species encoding Câ variants lacking the exon 4. When using this method, we also demonstrated that alternative splicing of the exon 4 probably does not occur in human peripheral blood leukocytes, despite the fact that they express the Câ1 and Câ2 variants. This implies that alternative splicing of the Câ exon 4 may only take place in nerve cells. The novel splice variants displayed no in vitro catalytic activity and did not induce the expression of a CRE-regulated reporter gene. Finally, we demonstrated that the Câ variants lacking the exon 4 were able to form PKA holoenzymes by associating with the RIIá subunit. However, these PKA holoenzymes did not dissociate, not even in the presence of high non-physiological levels of cAMP.