Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.     

AKAP-Lbc: a molecular scaffold for the integration of cyclic AMP and Rho transduction pathways

A Kinase-anchoring proteins (AKAPs) are a family of functionally related proteins involved in the targeting of the PKA holoenzyme towards specific physiological substrates. We have recently identified a novel anchoring protein expressed in cardiomyocytes, called AKAP-Lbc, that functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) that activates the GTPase RhoA. Here, we discuss the most recent findings elucidating the molecular mechanisms and the transduction pathways involved in the regulation of the AKAP-Lbc signaling complex inside cells. We could show that AKAP-Lbc is regulated in a bi-directional manner by signals that activate or deactivate its Rho-GEF activity. Activation of AKAP-Lbc occurs in response to agonists that stimulate G proteins coupled receptors linked to the heterotrimeric G protein G12, whereas inactivation occurs through mechanisms that require phosphorylation of AKAP-Lbc by anchored PKA and subsequent recruitment of the regulatory protein 14-3-3. Interestingly, we could demonstrate that AKAP-Lbc can form homo-oligomers inside cells and that 14-3-3 can inhibit the Rho-GEF activity of AKAP-Lbc only when the anchoring protein adopts an oligomeric conformation. These findings reveal the molecular architecture of the AKAP-Lbc transduction complex and provide a mechanistic explanation of how upstream signaling pathways can be integrated within the AKAP-Lbc transduction complex to precisely modulate the activation of Rho.     

Leucine zipper-mediated homo-oligomerization regulates the Rho-GEF activity of AKAP-Lbc

AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha-subunit of the heterotrimeric G protein G(12), whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family.     

Plugging PKA into ERK scaffolds

Cancers often arise in part through derangements in protein kinase signaling. A striking example of this is the finding that approximately 30% of human tumors have mutations in Ras or B-Raf, leading to aberrant ERK kinase activation. Kinase signaling networks are often organized by scaffolding and anchoring proteins that help shape the dynamics of signal processing. AKAP-Lbc associates with the ERK scaffold protein KSR-1 to organize a growth factor and cAMP responsive signaling network. AKAP-Lbc also directs PKA phosphorylation of KSR-1 on a critical residue to ensure maximal signaling efficiency.     

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.     

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.     

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

The role of protein kinase D in neurotensin secretion mediated by protein kinase C-alpha/-delta and Rho/Rho kinase

Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal (GI) secretion, motility, and growth as well as the proliferation of NT receptor positive cancers. Secretion of NT is regulated by phorbol ester-sensitive protein kinase C (PKC) isoforms-alpha and -delta and may involve protein kinase D (PKD). The purpose of our present study was: (i) to define the role of PKD in NT release from BON endocrine cells and (ii) to delineate the upstream signaling mechanisms mediating this effect. Here, we demonstrate that small interfering RNA (siRNA) targeted against PKD dramatically inhibited both basal and PMA-stimulated NT secretion; NT release is significantly increased by overexpression of PKD. PKC-alpha and -delta siRNA attenuated PKD activity, whereas overexpression of PKC-alpha and -delta enhanced PKD activity. Rho kinase (ROK) siRNA significantly inhibited NT secretion, whereas overexpression of ROKalpha effectively increased NT release. Rho protein inhibitor C3 dramatically inhibited both NT secretion and PKD activity. In conclusion, our results demonstrate that PKD activation plays a central role in NT peptide secretion; upstream regulators of PKD include PKC-alpha and -delta and Rho/ROK. Importantly, our results identify novel signaling pathways, which culminate in gut peptide release.     

A novel tyrosine phosphorylation site in protein kinase D contributes to oxidative stress-mediated activation

Protein kinase D1 (PKD1) is a mediator of oxidative stress signaling where it regulates cellular detoxification and survival. Critical for the regulation of PKD1 activity in response to oxidative stress are Src- and Abl-mediated tyrosine phosphorylations that eventually lead to protein kinase Cdelta (PKCdelta)-mediated activation of PKD1. Here we identify Tyr95 in PKD1 as a previously undescribed phosphorylation site that is regulated by oxidative stress. Our data suggest that PKD1 phosphorylation at Tyr95 generates a binding motif for PKCdelta, and that oxidative stress-mediated PKCdelta/PKD interaction results in PKD1 activation loop phosphorylation and activation. We further analyzed all PKD isoforms for this mechanism and show that PKD enzymes PKD1 and PKD2 are targets for PKCdelta in response to oxidative stress, and that PKD3 is not a target because it lacks the relevant tyrosine residue that generates a PKCdelta interaction motif.     

AKAP79 and the evolution of the AKAP model

A molecular explanation for the specificity of the cAMP-dependent protein kinase (PKA) can be provided by its compartmentalization through association with A-kinase-anchoring proteins (AKAPs). Structural and functional studies have led to the development of an anchoring model proposing that AKAPs contain a common PKA binding domain and a unique subcellular targeting domain. The discovery that AKAPs can bind other signaling enzymes led to the addition of a third property, that of scaffolding molecule. Recent research has now expanded the role of AKAPs to members of multiunit complexes containing both upstream activators and downstream targets.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Protein kinase D: a family affair

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.     

Regulation of type II PIP kinase by PKD phosphorylation

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.     

Cardiac function modulation depends on the A‐kinase anchoring protein complex

The A‐kinase anchoring proteins (AKAPs) are a group of structurally diverse proteins identified in various species and tissues. These proteins are able to anchor protein kinase and other signalling proteins to regulate cardiac function. Acting as a scaffold protein, AKAPs ensure specificity in signal transduction by enzymes close to their appropriate effectors and substrates. Over the decades, more than 70 different AKAPs have been discovered. Accumulative evidence indicates that AKAPs play crucial roles in the functional regulation of cardiac diseases, including cardiac hypertrophy, myofibre contractility dysfunction and arrhythmias. By anchoring different partner proteins (PKA, PKC, PKD and LTCCs), AKAPs take part in different regulatory pathways to function as regulators in the heart, and a damaged structure can influence the activities of these complexes. In this review, we highlight recent advances in AKAP‐associated protein complexes, focusing on local signalling events that are perturbed in cardiac diseases and their roles in interacting with ion channels and their regulatory molecules. These new findings suggest that AKAPs might have potential therapeutic value in patients with cardiac diseases, particularly malignant rhythm.     

Src Homology 2 Domain-containing Phosphatase 2 (Shp2) Is a Component of the A-kinase-anchoring Protein (AKAP)-Lbc Complex and Is Inhibited by Protein Kinase A (PKA) under Pathological Hypertrophic Conditions in the Heart

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy.     

Protein kinase D regulates cofilin activity through p21-activated kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.     

Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H₂O₂-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H₂O₂ treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.     

Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.