Cyclic AMP-dependent protein kinase A regulates the alternative splicing of CaMKIIδ

Abstract

Ca(2+)/calmodulin-dependent protein kinase (CaMK) IIδ is predominantly expressed in the heart. There are three isoforms of CaMKIIδ resulting from the alternative splicing of exons 14, 15, and 16 of its pre-mRNA, which is regulated by the splicing factor SF2/ASF. Inclusion of exons 15 and 16 or of exon 14 generates δA or δB isoform. The exclusion of all three exons gives rise to δC isoform, which is selectively increased in pressure-overload-induced hypertrophy. Overexpression of either δB or δC induces hypertrophy and heart failure, suggesting their specific role in the pathogenesis of hypertrophy and heart failure. It is well known that the β-adrenergic-cyclic AMP-dependent protein kinase A (PKA) pathway is implicated in heart failure. To determine the role of PKA in the alternative splicing of CaMKIIδ, we constructed mini-CaMKIIδ genes and used these genes to investigate the regulation of the alternative splicing of CaMKIIδ by PKA in cultured cells. We found that PKA promoted the exclusion of exons 14, 15, and 16 of CaMKIIδ, resulting in an increase in δC isoform. PKA interacted with and phosphorylated SF2/ASF, and enhanced SF2/ASF's activity to promote the exclusion of exons 14, 15, and 16 of CaMKIIδ, leading to a further increase in the expression of δC isoform. These findings suggest that abnormality in β-adrenergic-PKA signaling may contribute to cardiomyopathy and heart failure through dysregulation in the alternative splicing of CaMKIIδ exons 14, 15, and 16 and up-regulation of CaMKIIδC.

Figure 1. Alternative splicing of CaMKIIδ exons 14, 15, and 16 generates three splicing variants, corresponding to CaMKIIδ isoforms A, B, and C, respectively.

A and B, Schematic diagram of the alternative splicing of exons 14, 15, and 16 of mini -CaMKIIδ-genes, pCI/CaMKIIδ_E12–E17 (A) and pCI/CaMKIIδ_E13–E17 (B). C and D, Three splicing variants was generated from mini-CaMKIIδ gene, pCI/CaMKIIδ_E12–E17 (C) or pCI/CaMKIIδ_E13–E17 (D), after transfection into HEK-293T or COS7 cells, respectively, for 48 hrs. The total RNA was used for measurement of the splicing products with RT-PCR.

Figure 2. PKA promotes exclusion of exons 14, 15, and 16 of CaMKIIδ.

A, Forskolin treatment activated PKA. HEK-293T cells were transfected with pCI/CaMKIIδ_E13–E17 for 40 hrs and then treated with 10 µM forskolin for 8 hrs. The cells were subjected to Western blots for detection of PKA activity with anti-phosphorylated CREB at Ser133 and anti-CREB. B, Forskolin treatment promoted the exclusion of exons 14, 15, and 16, resulting in an increase in CaMKIIδC expression. HEK-293T cells were transfected with pCI/CaMKIIδ_E13–E17 for 40 hrs and then treated with 10 µM forskolin for 8 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry, and the percentage of each splicing form was calculated. C, Overexpression of PKA-Cα increased PKA activity. HEK-293T cells were co-transfected with pCI/CaMKIIδ_E12–E17 and pCI/PKA-Cα for 48 hrs. The PKA activity in the cells was measured by phosphorylation of CREB at Ser133 with Western blots. D, Overexpression of PKA-Cα promoted the exclusion of exons 14, 15, and 16 of CaMKII™. HEK-293T cells were cotransfected with pCI/CaMKIIδ_E13–E17 and pCI/PKA-Cα for 48 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry, and the percentage of each splicing form was calculated. The Data are presented as mean ± S.D. *p<0.05 versus control treatment.

Figure 3. SF2/ASF promotes the exclusion of exons 14, 15, and 16 of CaMKIIδ.

A and B, Overexpression of SF2/ASF promoted the exclusion of exons 14, 15, and 16 of pCI/CaMKIIδ_E12–E17 in COS7 cells. PCI/CAMKIIδ_E12–E17 was co-transfected with pCEP4/SF2/ASF into COS7 cells for 48 hr. The splicing products were measured with RT-PCR (A). Each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated (B). C and D, Overexpression of SF2/ASF promoted the exclusion of exons 14, 15, and 16 of pCI/CaMKIIδ_E13–E17 in HEK-239T. PCI/CAMKIIδ_E12–E17 was co-transfected with pCEP4/SF2/ASF into HEK-293T cells for 48 hr. The splicing products were measured with RT-PCR (C). The each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated (D). The Data are presented as mean ± S.D. *p<0.05 versus control treatment.

Figure 4. PKA phosphorylates and interacts with SF2/ASF.

A, Recombinant GST-SF2/ASF or GST was incubated with PKA in the presence of [γ-³²P]ATP at 30°C for 10 min, and the reaction mixture was then separated by SDS-PAGE and visualized with Coommassie blue staining (lower panel) or autoradiograph (upper panel). B, PKA-Cα was pull-down by SF2/ASF. GST-SF2/ASF or GST coupled onto glutathione-Sepharose or glutathione-Sepharose (GSH-beads) was incubated with rat brain extract. After extensively washing, the bound proteins were analyzed by Western blots developed with anti-GST or anti-PKA-Cα. C, PKA-Cα was co-immunoprecipitated by anti-HA. SF2/ASF tagged with HA were expressed in HEK-293FT cells for 48 h. The cell extracts were immunoprecipitated with anti-HA, and the immunoprecipitates were subjected to Western blots developed with anti-HA and anti-PKA-Cα. D, HeLa cells were transfected with pCEP4/SF2/ASF and treated without (Con) or with forskolin (Fors) for 30 min, followed by triple immunofluorescence staining.

Figure 5. PKA activation enhances SF2/ASF-promoted exclusion of exons 14, 15, and 16 of CaMKIIδ.

A, HEK-293FT cells were transfected with pCI/CaMKIIδ_E13–E17 for 40 hrs and then treated with 10 µM forskolin or 20 µM isoproterenol for 8 hrs. The splicing products were measured with RT-PCR. Each splicing product was quantitated by densitometry and the percentage of each splicing form was calculated. The Data are presented as mean ± S.D. *p<0.05 versus control treatment. B, Proposed mechanism by which abnormalities of β-adrenergic-PKA-pathway dysregulates the alternative splicing of exons 14, 15, and 16 of CaMKIIδ via SF2/ASF.