A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart

Stimulation of β-adrenergic receptors activates type I and II cyclic AMP–dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase–anchoring proteins (AKAPs) regulates cyclic AMP–dependent signaling in the cell. We investigated the expression and localization of AKAP100 in adult hearts. By immunoblotting, we identified AKAP100 in adult rat and human hearts, and showed that type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. By immunofluorescence and confocal microscopy of rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, we localized AKAP100 to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus α-actinin–specific antibodies or AKAP100 plus ryanodine receptor–specific antibodies, confocal images showed AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, was colocalized with AKAP100 in the rat heart. Our studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart.

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Subcellular Localization and Biological Actions of Activated RSK1 Are Determined by Its Interactions with Subunits of Cyclic AMP-Dependent Protein Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.01422-05 ◽

2006 ◽

Vol 26 (12) ◽

pp. 4586-4600 ◽

Cited By ~ 40

Author(s):

Deepti Chaturvedi ◽

Helen M. Poppleton ◽

Teresa Stringfield ◽

Ann Barbier ◽

Tarun B. Patel

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Nuclear Accumulation ◽

Regulatory Subunit ◽

Type I ◽

Dependent Protein Kinase ◽

Anchoring Proteins ◽

Dependent Protein ◽

Ribosomal S6 Kinase ◽

Biological Actions

ABSTRACT Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.

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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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Expression of cholinesterases and their anchoring proteins in rat heart

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2019-0565 ◽

2020 ◽

Vol 98 (7) ◽

pp. 473-476 ◽

Cited By ~ 1

Author(s):

Zuzana Kilianova ◽

Natalia Ciznarova ◽

Kristina Szmicsekova ◽

Lubica Slobodova ◽

Anna Hrabovska

Keyword(s):

Rat Heart ◽

Cholinergic System ◽

Ischemia Reperfusion ◽

Expression Patterns ◽

Molecular Forms ◽

Ach Release ◽

Anchoring Proteins ◽

Molecular Variants ◽

Anchoring Protein ◽

Membrane Anchoring

Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia–reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.

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