PKA‐binding domain of AKAP8 is essential for direct interaction with DPY30 protein

Abstract

The main function of the A kinase-anchoring proteins (AKAPs) is to target the cyclic AMP-dependent protein kinase A (PKA) to its cellular substrates through the interaction with its regulatory subunits. Besides anchoring of PKA, AKAP8 participates in regulating the histone H3 lysine 4 (H3K4) histone methyltransferase (HMT) complexes. It is also involved in DNA replication, apoptosis, transcriptional silencing of rRNA genes, alternative splicing, and chromatin condensation during mitosis. In this study, we focused on the interaction between AKAP8 and the core subunit of all known H3K4 HMT complexes-DPY30 protein. Here, we demonstrate that the PKA-binding domain of AKAP8 and the C-terminal domain of DPY30, also called Dpy-30 motif, are crucial for the interaction between these proteins. We show that a single amino acid substitution in DPY30 L69D affects its dimerization and completely abolishes its interaction with AKAP8 and another DPY30-binding partner brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), which is also AKAP domain-containing protein. We further demonstrate that AKAP8 interacts with DPY30 and the RII alpha regulatory subunit of PKA both in the interphase and in mitotic cells, and we show evidences that AKAP8L, a homologue of AKAP8, interacts with core subunits of the H3K4 HMT complexes, which suggests its role as a potential regulator of these complexes. The results presented here reinforce the analogy between AKAP8-RII alpha and AKAP8-DPY30 interactions, postulated before, and improve our understanding of the complexity of the cellular functions of the AKAP8 protein.Abbreviations AKAP8, A-kinase anchoring protein 8; AKAP8L, A-kinase anchoring protein 8 like; ASH2L, absent, small, or homeotic 2-like protein; BAP18, BPTF-associated protein of 18 kDa; BIG1, brefeldin A-inhibited guanine nucleotide-exchange protein; co-IP, co-immunoprecipitation; DBM, DPY30-binding motif; GST, glutathione S-transferase; H3K4, histone H3 lysine 4; HDAC3, histone deacetylase 3; HEK293, human embryonic kidney 293 cell line; His-tag, polyhistidine-tag; HMT, histone methyltransferase; Kif2A, kinesin family member 2A; KMT, histone lysine methyltransferase; MLL, mixed lineage leukemia; PI, propidium iodide; PKA, cAMP-dependent protein kinase; PRKAR2a, protein kinase cAMP-dependent type II regulatory subunit alpha; RbBP5, retinoblastoma-binding protein 5; RIIa, cAMP-dependent protein kinase type IIalpha regulatory subunit; SAC, spindle assembly checkpoint; TAP tag, tandem affinity purification tag; TPR, translocated promoter region protein; WB, Western blot; WDR5, WD repeat domain 5; WRAD, WDR5-RbBP5-ASH2L-DPY30 complex.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations

“…To date, very few of the aberrantly spliced isoforms identified in SFmut malignancies have been functionally characterized. 6 We identified aberrant splicing of the mitosis regulators SEPT2 99,100 and AKAP8, 71 leading to their downregulation in the CD34 1 cells of SF3B1mut and SRSF2mut MDS, respectively. Anemia is a hallmark of MDS, 1 and we found that knockdown of SEPT2 or AKAP8 in human hematopoietic progenitors resulted in markedly impaired erythroid cell growth and differentiation.…”

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The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes

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