• Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

      Willis, Brandon S.; Niswender, Colleen M.; Su, Thomas; Amieux, Paul S.; McKnight, G. Stanley; Mei, Lin; Department of Neurology (2011-04-12)
      We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any celltype that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIa regulatory subunit (RIaB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIaB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIaB demonstrated a reduction in PKA activity and inhibition of cAMPresponsive gene expression. Mice expressing RIaB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIaB allele in vivo provides a novel system for the analysis of PKA function in physiology.
    • Mutations in AKAP5 Disrupt Dendritic Signaling Complexes and Lead to Electrophysiological and Behavioral Phenotypes in Mice

      Weisenhaus, Michael; Allen, Margaret L.; Yang, Linghai; Lu, Yuan; Nichols, C. Blake; Su, Thomas; Hell, Johannes W.; McKnight, G. Stanley; Mei, Lin; Department of Neurology; et al. (2010-04-23)
      AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIa or RIIb as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and b-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.