Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: a disease susceptibility polymorphism

Abstract

The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity andor mortality. To test this assumption, we compared allele frequencies of 6,500 single-nucleotide polymorphisms (SNPs) located in approximately 5,000 genes between DNA pools of age-stratified healthy, European-American individuals. A SNP that results in an amino acid change from Ile to Val in the dual-specific A kinase-anchoring protein 2 (d-AKAP2) gene, showed the strongest correlation with age. Subsequent analysis of an independent sample indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardiogram PR interval. The IleVal SNP is located in the A-kinase-binding domain. An in vitro binding assay revealed that the Ile variant bound approximately 3-fold weaker to the protein kinase A (PKA)-RIalpha isoform than the Val variant. This decreased affinity resulted in alterations in the subcellular distribution of the recombinantly expressed PKA-RIalpha isoform. Our study suggests that alterations in PKA-RIalpha subcellular localization caused by variation in d-AKAP2 may have a negative health prognosis in the aging population, which may be related to cardiac dysfunction. Age-stratified samples appear to be useful for screening SNPs to identify functional gene variants that have an impact on health.

Figure 1

Neighbor-joining tree of genetic distances among the four European-American discovery groups (YF, YM, OF, and OM) and DNA pools from three additional ethnic groups. HI, Hispanic American; AF, African American; AS, Asian American; YF, young female; YM, young male; OF, old female; OM, old male. The lengths of branches are proportional to the pairwise genetic distances between pools.

Figure 2

Characterization of the Ile/Val variant in the AKB domain of d-AKAP2. (a) Sequence alignment of the highly conserved AKB domain from several species. The residues comprising the hydrophobic face of the helix are highlighted as black boxes. The variable position I646V is part of the hydrophobic face (arrow). (b) Pull-down assays using the C-terminal, AKB-containing portion of d-AKAP2 fused to GST of mouse (Mo) and human (Hu). The Val variants at position 24 (V24) showed higher affinity to RIα as compared with the Ile forms (I24). Equal levels of GST-AKB proteins were estimated by SDS gel electrophoresis. An excess of GST-AKB was used so that the amount of R subunit bound to the glutathione beads reflects the binding affinities. Two separate experiments were performed to verify that GST-AKB proteins are in excess: when half the amount of GST-AKB was used, a similar amount of the R subunit was pulled down; when half of the amount of R protein was used, approximately half as much was bound. RIα and RIIα were used at concentrations of 2 and 20 μM, respectively. (c) Quantitative binding assay using a fluorescently labeled peptide of both the AKB variants. Binding of RIα to both the Ile (■) and Val (▴) variants is illustrated. The Val allele results in an increase in binding to the RIα isoform. The curves through the data are a fit to a 1:1 binding model. A deletion mutant of RIα (▵ 1–91) (♦), which lacks the AKAP binding domain, does not bind to either peptide. (Inset) The dissociation constants for the regulatory subunit isoforms.

Figure 3

Association of the AKB domain of d-AKAP2 with RIα and RIIα in 10T(1/2) cells. Through fusion with a mitochondrial targeting domain, the AKB domain directs different amounts of RI and RII to the mitochondria. The Val variant (V24) effectively binds RIα (a–c) and targets RIα-D/D-GFP to mitochondria (b). The Ile variant (I24) binds to RIα less effectively (d–f) and appears to be distributed evenly in the cells (e). In contrast, both variants show no apparent differences for RIIα (g–l) binding; they all colocalize at mitochondria (h and k).