Lorene K. Langeberg

Coordination of Three Signaling Enzymes by AKAP79, a Mammalian Scaffold Protein

Multivalent binding proteins, such as the yeast scaffold protein Sterile-5, coordinate the location of kinases by serving as platforms for the assembly of signaling units. Similarly, in mammalian cells the cyclic adenosine 3′,5′-monophosphate-dependent protein kinase (PKA) and phosphatase 2B [calcineurin (CaN)] are complexed by an A kinase anchoring protein, AKAP79. Deletion analysis and binding studies demonstrate that a third enzyme, protein kinase C (PKC), binds AKAP79 at a site distinct from those bound by PKA or CaN. The subcellular distributions of PKC and AKAP79 were similar in neurons. Thus, AKAP79 appears to function as a scaffold protein for three multifunctional enzymes.

The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways.

Cyclic adenosine 3′, 5′-monophosphate (cAMP) is a ubiquitous mediator of intracellular signalling events. It acts principally through stimulation of cAMP-dependent protein kinases (PKAs) but also activates certain ion channels and guanine nucleotide exchange factors (Epacs). Metabolism of cAMP is catalysed by phosphodiesterases (PDEs). Here we identify a cAMP-responsive signalling complex maintained by the muscle-specific A-kinase anchoring protein (mAKAP) that includes PKA, PDE4D3 and Epac1. These intermolecular interactions facilitate the dissemination of distinct cAMP signals through each effector protein. Anchored PKA stimulates PDE4D3 to reduce local cAMP concentrations, whereas an mAKAP-associated ERK5 kinase module suppresses PDE4D3. PDE4D3 also functions as an adaptor protein that recruits Epac1, an exchange factor for the small GTPase Rap1, to enable cAMP-dependent attenuation of ERK5. Pharmacological and molecular manipulations of the mAKAP complex show that anchored ERK5 can induce cardiomyocyte hypertrophy. Thus, two coupled cAMP-dependent feedback loops are coordinated within the context of the mAKAP complex, suggesting that local control of cAMP signalling by AKAP proteins is more intricate than previously appreciated.

Ubiquitination regulates PSD-95 degradation and AMPA receptor surface expression

PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites by proteasome-dependent degradation. Mutations that block PSD-95 ubiquitination prevent NMDA-induced AMPA receptor endocytosis. Likewise, proteasome inhibitors prevent NMDA-induced AMPA receptor internalization and synaptically induced long-term depression. This is consistent with the notion that PSD-95 levels are an important determinant of AMPA receptor number at the synapse. These data suggest that ubiquitination of PSD-95 through an Mdm2-mediated pathway is critical in regulating AMPA receptor surface expression during synaptic plasticity.

mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module

Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle‐selective A‐kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram‐inhibited cAMP‐specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP‐associated phosphodiesterase activity. Disruption of PKA–mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

BACKGROUND 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low‐molecular weight A‐kinase Anchoring Protein, called AKAP18, which targets the cAMP‐dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L‐type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid‐modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK‐293 cells expressing the cardiac L‐type Ca2+ channel promoted a 34 9% increase in cAMP‐responsive Ca2+ currents. In contrast, a targeting‐deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP‐1‐mediated insulin secretion in a pancreatic β cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP‐responsive events that may involve ion channel activation.

Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein.

BACKGROUND Subcellular targeting of protein kinases and phosphatases provides a mechanism for co-localizing these enzymes with their preferred substrates. A recently identified mammalian scaffold protein, AKAP79, controls the location of two broad-specificity kinases and a phosphatase. RESULTS We have identified and characterized another mammalian scaffold protein which coordinates the location of protein kinase A and protein kinase C. We isolated a cDNA encoding a 250 kDa A-skinase anchoring protein (AKAP) called gravin, which was originally identified as a cytoplasmic antigen recognized by myasthenia gravis sera. Sequence homology to proteins that are known to bind protein kinase C suggests that gravin also binds this kinase. Studies of binding in vitro show that residues 1526-1780 of gravin bind the regulatory subunit (RII) of protein kinase A with high affinity, and residues 265-556 bind protein kinase C. Gravin expression in human erythroleukemia cells can be induced with phorbol ester, resulting in the detection of a 250 kDa RII- and PKC-binding protein. Immunolocalization experiments show that gravin is concentrated at the cell periphery and is enriched in filopodia. Gravin staining is coincident with an AKAP detected by an in situ RII-overlay assay, and a PKA-gravin complex can be isolated from human erythroleukemia cells. CONCLUSIONS We present biochemical evidence that gravin forms part of a signaling scaffold, and propose that protein kinases A and C may participate in the coordination of signal transduction events in the filopodia of human erythroleukemia cells.

Scar/WAVE‐1, a Wiskott–Aldrich syndrome protein, assembles an actin‐associated multi‐kinase scaffold

WAVE proteins are members of the Wiskott–Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho‐related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE‐1 in a screen for rat brain A kinase‐anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE‐1 interacts with cAMP‐dependent protein kinase (PKA) and Abl kinases when expressed in HEK‐293 cells, and both enzymes co‐purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA–WAVE‐1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE‐1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet‐derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE‐1 as an actin‐associated scaffolding protein that recruits PKA and Abl.

AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists

M-type (KCNQ2/3) potassium channels are suppressed by activation of Gq/11-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(ΔA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(ΔA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.