The structure and expression of cyclic AMP-dependent protein kinase subunit genes in Caenorhabditis elegans and mouse Friend erythroleukemic cells

Abstract

cAMP-dependent protein kinase (PKA) regulates numerous cellular processes in eukaryotes in response to extracellular and intracellular signals, via a flexible post-translational modification system. Phosphorylation of substrate proteins leads to control of enzyme activity and/or gene expression. PKA is a heterotetramer composed of two regulatory (R) subunits, present as a homodimer, and two catalytic (C) subunits. The special properties of PKA isozymes are conferred by distinct R subunits: RI{dollar}\alpha{dollar}, RI{dollar}\beta{dollar}, RII{dollar}\alpha{dollar} and RII{dollar}\beta{dollar}. Two C subunit isoforms have been characterized: C{dollar}\alpha{dollar} and C{dollar}\beta{dollar}. The R subunit isoforms may adapt PKA for specialized functions in the tissues in which they are expressed. This thesis addresses the control of the expression of R and C isoforms in two model systems with unique advantages for the study of developmental, differentiation and hormonal control of gene expression.;Friend erythroleukemic cells contain RI{dollar}\alpha{dollar}, two RII subunits, and C{dollar}\alpha{dollar}. Previous work in this laboratory demonstrated that when Friend cells are subjected to agents that chronically elevate cAMP levels (e.g. forskolin), the amount and rate of synthesis of RII52 selectively increases 10-15-fold. When the cells are induced to differentiate, similar increases in RII52 are accompanied by increases in total PKA activity. This thesis will establish the identity of RII52 with RII{dollar}\beta{dollar}, and show that elevated cAMP levels are associated with a 25-30-fold increase in the abundance of RII{dollar}\beta{dollar} mRNA. Surprisingly, the rate of transcriptional initiation of the RII{dollar}\beta{dollar} gene and the stability of the RII{dollar}\beta{dollar} mRNA are unaffected. Rather, two precursors of mature RII{dollar}\beta{dollar} mRNA accumulate in the nucleus of Friend cells treated with forskolin. This post-transcriptional effect is the direct result of PKA C subunit activity, since it is reproduced in Friend cells that overexpress C from an expression vector under the control of a metallothionein promoter. In differentiated Friend cells, RII{dollar}\beta{dollar} and C{dollar}\alpha{dollar} mRNAs accumulate in the absence of an elevation in the rate of transcriptional initiation of these genes.;The nematode C. elegans provides many advantages for a molecular genetic approach to signal transduction. This thesis describes the characterization of PKA subunits and their mRNAs and genes in C. elegans. Only one type of R cDNA could be found, corresponding to RI{dollar}\alpha{dollar}. A C cDNA was characterized that encodes a C subunit that is highly (82%) identical to mammalian C subunits. A novel cDNA was also discovered that corresponds to mRNA that is the product of an alternative splicing pathway of C transcripts. The novel mRNA predicts a C subunit with a unique carboxyl-terminus relative to previously described C subunits. Evidence is presented for the presence of this protein in the nematode. The C transcript is trans-spliced to the ubiquitous 22 bp SL-1 leader sequence.