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Regulation of the interactions between human eIF5 and eIF1A by the CK2 kinase

ABSTRACT: Translation initiation in eukaryotes relies on a complex network of interactions that are continuously reorganized throughout the process. As more information becomes available about the structure of the ribosomal preinitiation complex (PIC) at various points in translation initiation, new questions arise about which interactions occur when, their roles, and regulation. The eukaryotic translation factor (eIF) 5 is the GTPase-activating protein (GAP) for the GTPase eIF2, which brings the initiator Met-tRNAi to the PIC. eIF5 also plays a central role in PIC assembly and remodeling through interactions with other proteins, including eIFs 1, 1A, and 3c. Phosphorylation by casein kinase 2 (CK2) significantly increases the eIF5 affinity for eIF2. The interaction between eIF5 and eIF1A was reported to be mediated by the eIF5 C-terminal domain (CTD) and the eIF1A N-terminal tail. Here, we report a new contact interface, between eIF5-CTD and the oligonucleotide/oligosaccharide-binding fold (OB) domain of eIF1A, which contributes to the overall affinity between the two proteins. We also show that the interaction is modulated by dynamic intramolecular interactions within both eIF5 and eIF1A. CK2 phosphorylation of eIF5 increases its affinity for eIF1A, offering new insights into the mechanisms by which CK2 stimulates protein synthesis and cell proliferation. Graphical abstract Image 1 Highlights • eIF5-CTD interacts with both the N-terminal tail and the OB domain of eIF1A.• The OB domain contacts stabilize the overall interaction.• The eIF1A C-terminal tail and the eIF5 DWEAR motif interfere with OB domain binding.• CK2 phosphorylation of eIF5 increases its affinity for eIF1A.

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PROVIDER: S-EPMC9508154 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Project description:CK2, a serine/threonine (Ser/Thr) kinase present in eukaryotic cells, is known to have a vast number of substrates. We have recently shown that it localizes to nuclei and at pores between hyphal compartments in Magnaporthe oryzae We performed a pulldown proteomics analysis of M. oryzae CK2 catalytic subunit MoCKa to detect interacting proteins. The MoCKa pulldown was enriched for septum and nucleolus proteins and intrinsically disordered proteins (IDPs) containing a CK2 phosphorylation motif that is proposed to destabilize and unfold α-helices. This points to a function for CK2 phosphorylation and corresponding phosphatase dephosphorylation in the formation of functional protein-protein aggregates and protein-RNA/DNA binding. To test this as widely as possible, we used secondary data downloaded from databases from a large range of M. oryzae experiments, as well as data for a relatively closely related plant-pathogenic fungus, Fusarium graminearum We found that CKa expression was strongly positively correlated with Ser/Thr phosphatases, as well as with disaggregases (HSP104, YDJ1, and SSA1) and an autophagy-indicating protein (ATG8). The latter points to increased protein aggregate formation at high levels of CKa expression. Our results suggest a general role for CK2 in chaperoning aggregation and disaggregation of IDPs and their binding to proteins, DNA, and RNA.IMPORTANCE CK2 is a eukaryotic conserved kinase enzyme complex that phosphorylates proteins. CK2 is known to phosphorylate a large number of proteins and is constitutively active, and thus a "normal" role for a kinase in a signaling cascade might not be the case for CK2. Previous results on localization and indications from the literature point to a function for CK2 phosphorylation in shaping and folding of proteins, especially intrinsically disordered proteins, which constitute about 30% of eukaryotic proteins. We used pulldown of interacting proteins and data downloaded from a large range of transcriptomic experiments in M. oryzae and complemented these with data downloaded from a large range of transcriptomic experiments in Fusarium graminearum We found support for a general role for CK2 in aggregation and disaggregation of IDPs and their binding to proteins, DNA, and RNA-interactions that could explain the importance of CK2 in eukaryotic cell function and disease.

Project description:BackgroundProtein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (alpha) and two identical regulatory (beta) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2alpha in isolation. The present work explores the structural basis of this feature using computational analysis and modeling.ResultsWe have initially built a model of PKCK2alpha bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in beta turn, alpha helix, or 3(10) helices. This type of conformation is observed and accommodated well by PKCK2alpha in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2alpha phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2alpha complex. PKCK2beta undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme.ConclusionCharge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2alpha, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme.

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Regulation of the interactions between human eIF5 and eIF1A by the CK2 kinase

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