Increased Basal cAMP-dependent Protein Kinase Activity Inhibits the Formation of Mesoderm-derived Structures in the Developing Mouse Embryo

Abstract

A targeted disruption of the RI␣ isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RI␣ is the only isoform that is essential for early embryonic development. RI␣ homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RI␣ mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RI␣ mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RI␣ mutants to mice carrying a targeted disruption in the C␣ catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RI␣ is responsible for the phenotype. Primary embryonic fibroblasts from RI␣ mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

“…Perhaps the most definitive proof comes from data presented in Fig. 4D showing that SKIP cannot anchor PKA in RIα −∕− mouse embryonic fibroblasts (41). This genetic evidence augments cell-based support in Fig.…”

“…A rigorous genetic test of our hypothesis that SKIP is an exclusive RI anchoring protein was conducted in mouse embryonic fibroblasts (MEF) from RIα −∕− mice. These cells do not express RIα and thus are devoid of type I PKA (41). Initially, wild-type MEFs expressing FLAG-SKIP or FLAG-AKAP79 were generated, and FLAG immune complexes were assessed for copurification of PKA subunits by Western blotting and kinase activity (Fig.…”

Mys Protein Regulates Protein Kinase A Activity by Interacting with Regulatory Type Iα Subunit during Vertebrate Development

“…4, G and H). Although basal PKA activity is substantially increased in Prkar1a-deficient mouse embryos, which is consistent with our present results, a large amount of PKA responsive to cAMP is still retained and this PKA is thought to be inactivated by Prkar2a expressed in the mutant embryos (45). The results obtained in mice suggest that Prkar1a-deficient zebrafish embryos retain additional inactive PKA consisting of other regulatory subunits, such as Prkar2a.…”

Comparative PRKAR1A genotype–phenotype analyses in humans with Carney complex and prkar1a haploinsufficient mice

“…Prkar1a ϩ/Ϫ mice were generated by homologous recombination (12) and bred and studied per Cornell guidelines. M-mode and 2D echocardiography were performed with an Acuson (Mountain View, CA) Sequoia C256 and 15L8 probe.…”

“…To further assess consequences of PRKAR1A haploinsufficiency, we studied prkar1a ϩ/Ϫ mice (12). Although prkar1a Ϫ/Ϫ mice die with embryonic defects, prkar1a ϩ/Ϫ mice undergo normal embryogenesis and lack malformations (12,18).…”