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An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi.

ABSTRACT: KDEL receptor (KDELR) is a key protein that recycles escaped endoplasmic reticulum (ER) resident proteins from the Golgi apparatus back to the ER and maintains a dynamic balance between these two organelles in the early secretory pathway. Studies have shown that this retrograde transport pathway is partly regulated by two KDELR-interacting proteins, acyl-CoA-binding domain-containing 3 (ACBD3), and cyclic AMP-dependent protein kinase A (PKA). However, whether Golgi-localized ACBD3, which was first discovered as a PKA-anchoring protein in mitochondria, directly interacts with PKA at the Golgi and coordinates its signaling in Golgi-to-ER traffic has remained unclear. In this study, we showed that the GOLD domain of ACBD3 directly interacts with the regulatory subunit II (RII) of PKA and effectively recruits PKA holoenzyme to the Golgi. Forward trafficking of proteins from the ER triggers activation of PKA by releasing the catalytic subunit from RII. Furthermore, we determined that depletion of ACBD3 reduces the Golgi fraction of RII, resulting in moderate, but constitutive activation of PKA and KDELR retrograde transport, independent of cargo influx from the ER. Taken together, these data demonstrate that ACBD3 coordinates the protein secretory pathway at the Golgi by facilitating KDELR/PKA-containing protein complex formation.

SUBMITTER: Jia J

PROVIDER: S-EPMC10197108 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi.

Jia Jie J Tang Shuocheng S Yue Xihua X Jing Shuaiyang S Zhu Lianhui L Tan Chuanting C Gao Jingkai J Du Yulei Y Lee Intaek I Qian Yi Y

The Journal of biological chemistry 20230410 5

KDEL receptor (KDELR) is a key protein that recycles escaped endoplasmic reticulum (ER) resident proteins from the Golgi apparatus back to the ER and maintains a dynamic balance between these two organelles in the early secretory pathway. Studies have shown that this retrograde transport pathway is partly regulated by two KDELR-interacting proteins, acyl-CoA-binding domain-containing 3 (ACBD3), and cyclic AMP-dependent protein kinase A (PKA). However, whether Golgi-localized ACBD3, which was fir ...[more]

PMID: 37044218

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Project description:BackgroundThe asexual intra-erythrocytic multiplication of the malaria parasite Plasmodium falciparum is regulated by various molecular mechanisms. In eukaryotic cells, protein kinases are known to play key roles in cell cycle regulation and signaling pathways. The activity of cAMP-dependent protein kinase (PKA) depends on A-kinase anchoring proteins (AKAPs) through protein interactions. While several components of the cAMP dependent pathway-including the PKA catalytic and regulatory subunits-have been characterized in P. falciparum, whether AKAPs are involved in this pathway remains unclear. Here, PfAKAL, an open reading frame of a potential AKAP-like protein in the P. falciparum genome was identified, and its protein partners and putative cellular functions characterized.MethodsThe expression of PfAKAL throughout the erythrocytic cycle of the 3D7 strain was assessed by RT-qPCR and the presence of the corresponding protein by immunofluorescence assays. In order to study physical interactions between PfAKAL and other proteins, pull down experiments were performed using a recombinant PfAKAL protein and parasite protein extracts, or with recombinant proteins. These interactions were also tested by combining biochemical and proteomic approaches. As phosphorylation could be involved in the regulation of protein complexes, both PfAKAL and Pf14-3-3I phosphorylation was studied using a radiolabel kinase activity assay. Finally, to identify a potential function of the protein, PfAKAL sequence was aligned and structurally modeled, revealing a conserved nucleotide-binding pocket; confirmed by qualitative nucleotide binding experiments.ResultsPfAKAL is the first AKAP-like protein in P. falciparum to be identified, and shares 23 % sequence identity with the central domain of human AKAP18δ. PfAKAL is expressed in mature asexual stages, merozoites and gametocytes. In spite of homology to AKAP18, biochemical and immunochemical analyses demonstrated that PfAKAL does not interact directly with the P. falciparum PKA regulatory subunit (PfPKA-R), but instead binds and colocalizes with Pf14-3-3I, which in turn interacts with PfPKA-R. In vivo, these different interactions could be regulated by phosphorylation, as PfPKA-R and Pf14-3-3I, but not PfAKAL, are phosphorylated in vitro by PKA. Interestingly, PfAKAL binds nucleotides such as AMP and cAMP, suggesting that this protein may be involved in the AMP-activated protein kinase (AMPK) pathway, or associated with phosphodiesterase activities.ConclusionPfAKAL is an atypical AKAP that shares common features with human AKAP18, such as nucleotides binding. The interaction of PfAKAL with PfPKA-R could be indirectly mediated through a join interaction with Pf14-3-3I. Therefore, PfPKA localization could not depend on PfAKAL, but rather involves other partners.

Project description:We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K(d), 10 nm) for the type II regulatory subunit of protein kinase A (PKA RII). Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes. It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane. In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTPgammaS and prevented by brefeldin A. Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding. These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat. A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues. Neurobeachin, BGL, and approximately 10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family. The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome). Neurobeachin's subcellular localization, its coat protein-like membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates.

Project description:The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIβ. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIβ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIβ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that eliminate PI4KIIIβ association sensitized Aichi virus to PIK93, suggesting that disruption of the 3A/ACBD3/PI4KIIIβ complex may represent a novel target for therapeutic intervention that would be complementary to the inhibition of the kinase activity itself.

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An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi.

Publications

An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi.

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