Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins

A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

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NH2-Terminal Targeting Motifs Direct Dual Specificity A-Kinase–anchoring Protein 1 (D-AKAP1) to Either Mitochondria or Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.145.5.951 ◽

1999 ◽

Vol 145 (5) ◽

pp. 951-959 ◽

Cited By ~ 118

Author(s):

Lily Jun-shen Huang ◽

Lin Wang ◽

Yuliang Ma ◽

Kyle Durick ◽

Guy Perkins ◽

...

Keyword(s):

Splice Variants ◽

Type I ◽

Mitochondrial Targeting ◽

Regulatory Subunits ◽

Anchoring Proteins ◽

Dual Specificity ◽

Anchoring Protein ◽

Er Targeting ◽

Additional Level ◽

Necessary And Sufficient

Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase–anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (Huang et al., 1997. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. Deletion of N0(1-30) abolished mitochondrial targeting while N0(1-30)-GFP localized to mitochondria. Residues 1–30 of N0 are therefore necessary and sufficient for mitochondria targeting. Addition of the 33 residues of N1 targets D-AKAP1 to the ER and residues 1–63 fused to GFP are necessary and sufficient for ER targeting. Residues 14–33 of N1 are especially important for targeting to ER; however, residues 1–33 alone fused to GFP gave a diffuse distribution. N1(14-33) thus serves two functions: (a) it suppresses the mitochondrial-targeting motif located within residues 1–30 of N0 and (b) it exposes an ER-targeting motif that is at least partially contained within the N0(1-30) motif. This represents the first example of a differentially targeted AKAP and adds an additional level of complexity to the PKA signaling network.

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Regulation of Second Messenger Signaling in Hypoxic Neonatal Rats: Effect of Glucose, Oxygen and Epinephrine Resuscitation

International Conference on Earth, Environment and Life sciences (EELS-2014) Dec. 23-24, 2014 Dubai (UAE) ◽

10.15242/iicbe.c1214035 ◽

2014 ◽

Keyword(s):

Second Messenger ◽

Neonatal Rats ◽

Second Messenger Signaling ◽

Effect Of Glucose

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Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

Journal of Virology ◽

10.1128/jvi.73.5.3737-3743.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3737-3743 ◽

Cited By ~ 46

Author(s):

Alfred Klausegger ◽

Birgit Strobl ◽

Gerhard Regl ◽

Alexandra Kaser ◽

Willem Luytjes ◽

...

Keyword(s):

Substrate Specificity ◽

Solid Phase ◽

Hepatitis Virus ◽

Bovine Brain ◽

Brain Extract ◽

Binding Assays ◽

Bovine Coronavirus ◽

Close Relationship ◽

Influenza C Virus ◽

Cloned Gene

ABSTRACT We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substratesp-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

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Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

Biochemical Journal ◽

10.1042/bj20080234 ◽

2008 ◽

Vol 415 (1) ◽

pp. 27-33 ◽

Cited By ~ 45

Author(s):

Meghna Thakur ◽

Pradip K. Chakraborti

Keyword(s):

Protein Kinases ◽

Solid Phase ◽

Morphological Changes ◽

Binding Assays ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Peptidoglycan Biosynthesis ◽

Vitro Binding ◽

Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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Spatial organization enhances versatility and specificity in cyclic di-GMP signaling

Biological Chemistry ◽

10.1515/hsz-2020-0202 ◽

2020 ◽

Vol 401 (12) ◽

pp. 1323-1334

Author(s):

Sandra Kunz ◽

Peter L. Graumann

Keyword(s):

Protein Interactions ◽

Cell Cycle Progression ◽

Spatial Organization ◽

Caulobacter Crescentus ◽

Second Messenger ◽

Protein Protein Interactions ◽

Spatial Cues ◽

Signaling Specificity ◽

Regulatory Circuits ◽

Second Messenger Signaling

AbstractThe second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

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Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

Journal of Cell Science ◽

10.1242/jcs.113.13.2471 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2471-2483 ◽

Cited By ~ 3

Author(s):

I. Hofmann ◽

C. Mertens ◽

M. Brettel ◽

V. Nimmrich ◽

M. Schnolzer ◽

...

Keyword(s):

Epithelial Cells ◽

Intermediate Filament ◽

Solid Phase ◽

Specific Interaction ◽

Binding Assays ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Vitro Analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

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Fibronectin enhances Campylobacter fetus interaction with extracellular matrix components and INT 407 cells

Canadian Journal of Microbiology ◽

10.1139/w07-115 ◽

2008 ◽

Vol 54 (1) ◽

pp. 37-47 ◽

Cited By ~ 12

Author(s):

L. L. Graham ◽

T. Friel ◽

R. L. Woodman

Keyword(s):

Extracellular Matrix ◽

Solid Phase ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Bacterial Attachment ◽

Binding Assays ◽

Rgd Sequence ◽

Campylobacter Fetus ◽

Extracellular Matrix Components

Campylobacter fetus is a recognized pathogen of cattle and sheep that can also infect humans. No adhesins specific for C. fetus have to date been identified; however, bacterial attachment is essential to establish an infecting population. Scanning electron microscopy revealed C. fetus attachment to the serosal surface of human colonic biopsy explants, a location consistent with the presence of the extracellular matrix (ECM). To determine whether the ECM mediated C. fetus adherence, 7 C. fetus strains were assessed in a solid-phase binding assay for their ability to bind to immobilized ECM components. Of the ECM components assayed, adherence to fibronectin was noted for all strains. Attachment to ECM components was neither correlated with S-layer expression nor with cell-surface hydrophobicity. Ligand immunoblots, however, identified the S-layer protein as a major site of fibronectin binding, and modified ECM binding assays revealed that soluble fibronectin significantly enhanced the attachment of S-layer-expressing C. fetus strains to other ECM components. Soluble fibronectin also increased C. fetus adherence to INT 407 cells. This adherence was inhibited when INT 407 cells were incubated with synthetic peptides containing an RGD sequence, indicating that integrin receptors were involved in fibronectin-mediated attachment. Together, this data suggests that C. fetus can bind to immobilized fibronectin and use soluble fibronectin to enhance attachment to other ECM components and intestinal epithelial cells. In vivo, fibronectin would promote bacterial adherence, thereby, contributing to the initial interaction of C. fetus with mucosal and submucosal surfaces.

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