Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation.
It was once thought that proteins needed to have structures that were both ordered and stable, but this view was changed by the discovery that certain proteins contain regions that are disordered and flexible. In some cases these regions of intrinsic disorder help the protein to function by linking more stable regions that are active. However, in other proteins the disordered regions are themselves biologically active and can, for example, function as enzymes.
Protein kinase A is a family of enzymes that contains both ordered and disordered regions, with the ordered sections being involved in phosphorylation, a chemical process that is widely used for communication within cells. However, in order to initiate phosphorylation, these kinases must be anchored to a rigid substrate nearby, so a second group of proteins called AKAPs–which is short for A-kinase anchoring proteins–hold the kinases in place by binding to their disordered regions. These AKAPs also help the kinases to dock with other molecules involved in phosphorylation.
A full structural picture of how the kinases induce phosphorylation has yet to be obtained, partly because it is extremely difficult to determine the structure of the disordered regions within the kinases. Moreover, the AKAPs are also disordered, which makes it difficult to work out how the kinases are held in position.
Smith, Reichow et al. have used electron microscopy to reveal that the disordered region has two important roles: it determines how far away from the anchoring protein that the active region of the kinase can operate, and it influences how efficiently the kinase can bind to its target molecule in order to induce phosphorylation. Future challenges include investigating how the inherent flexibility of AKAP complexes contribute to the efficient phosphorylation of physiological targets.
A-kinase anchoring protein (AKAP); cAMP signaling; single particle reconstruction; cAMP-dependent kinase (PKA); electron microscopy; intrinsic disorder; None
The scaffolding protein WAVE-1 (Wiskott-Aldrich syndrome protein family member 1) directs signals from the GTPase Rac through the Arp2/3 complex to facilitate neuronal actin remodeling. The WAVE-associated GTPase activating protein called WRP is implicated in human mental retardation, and WAVE-1 knock-out mice have altered behavior. Neuronal time-lapse imaging, behavioral analyses, and electrophysiological recordings from genetically modified mice were used to show that WAVE-1 signaling complexes control aspects of neuronal morphogenesis and synaptic plasticity. Gene targeting experiments in mice demonstrate that WRP anchoring to WAVE-1 is a homeostatic mechanism that contributes to neuronal development and the fidelity of synaptic connectivity. This implies that signaling through WAVE-1 complexes is essential for neural plasticity and cognitive behavior.
WAVE-1; WRP; actin; Arp2/3; dendritic spine; synaptic plasticity
Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1–/– mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.
A kinase-anchoring proteins (AKAPs) organize compartmentalized pools of Protein Kinase A (PKA) to enable localized signaling events within neurons. However, it is unclear which of the many expressed AKAPs in neurons target PKA to signaling complexes important for long-lasting forms of synaptic plasticity and memory storage. In the forebrain, the anchoring protein gravin recruits a signaling complex containing PKA, PKC, calmodulin, and PDE4D to the β2-adrenergic receptor. Here, we show that mice lacking the α-isoform of gravin have deficits in PKA-dependent long-lasting forms of hippocampal synaptic plasticity including β2-adrenergic receptor-mediated plasticity, and selective impairments of long-term memory storage. Further, both hippocampal β2-adrenergic receptor phosphorylation by PKA, and learning-induced activation of ERK, are attenuated in the CA1 region of the hippocampus in mice lacking gravin-α. We conclude that gravin compartmentalizes a significant pool of PKA that regulates learning-induced β2-adrenergic receptor signaling and ERK activation in the hippocampus in vivo, organizing molecular interactions between glutamatergic and noradrenergic signaling pathways for long-lasting synaptic plasticity, and memory storage.
A-kinase anchoring proteins (AKAPs) are signaling scaffolds that contribute to various aspects of cAMP signaling. They do this by tethering protein kinase-A to specific subcellular sites, thereby focusing its activity toward relevant substrates. Recently the structural basis for these protein–protein interactions has been elucidated by x-ray crystallography. Recent reports have identified AKAPs that bind to adenylyl cyclases to regulate cAMP synthesis and that sequester phosphodiesterases to break down this second messenger locally. Another emerging aspect of AKAP function is their role in integrating cAMP signaling with other signaling pathways. For example, molecular and genetic approaches have been used to show that the neuronal anchoring protein WAVE1 integrates signaling from PKA and Cdk5 to regulate actin polymerization and cytoskeletal events.
The IL28B genotype is a critical determinant of interferon response in patients infected with hepatitis C virus genotype 1. We describe an allele-specific PCR assay for the IL28B genotype. The assay is simple and robust, uses commonly available real-time PCR instrumentation, and is well suited for clinical laboratories offering IL28B genotyping.
General population screening for hepatitis C virus (HCV) is likely to be a cost-effective approach to detect undiagnosed HCV in the United States, but a broad initiative aimed at improving referral and treatment would be necessary to substantially reduce population-level health outcomes.
(See the Editorial Commentary by Deuffic-Burban and Yazdanpanah, on pages 1272–4.)
Background. Current US guidelines recommend limiting hepatitis C virus (HCV) screening to high-risk individuals, and 50%–75% of infected persons remain unaware of their status.
Methods. To estimate the cost-effectiveness and population-level impact of adding one-time HCV screening of US population aged 20–69 years to current guidelines, we developed a decision analytic model for the screening intervention and Markov model with annual transitions to estimate natural history. Subanalyses included protease inhibitor therapy and screening those at highest risk of infection (birth year 1945–1965). We relied on published literature and took a lifetime, societal perspective.
Results. Compared to current guidelines, incremental cost per quality-adjusted life year gained (ICER) was $7900 for general population screening and $4200 for screening by birth year, which dominated general population screening if cost, clinician uptake, and median age of diagnoses were assumed equivalent. General population screening remained cost-effective in all one-way sensitivity analyses, 30 000 Monte Carlo simulations, and scenarios in which background mortality was doubled, all genotype 1 patients were treated with protease inhibitors, and most parameters were set unfavorable to increased screening. ICER was lowest if screening was applied to a population with liver fibrosis similar to 2010 estimates. Approximately 1% of liver-related deaths would be averted per 15% of the general population screened; the impact would be greater with improved referral, treatment uptake, and cure.
Conclusions. Broader screening for HCV would likely be cost-effective, but significantly reducing HCV-related morbidity and mortality would also require improved rates of referral, treatment, and cure.
L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of CaV1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.1 Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Cav1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of CaV1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of CaV1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Cav1.2 intracellular regions.
calcium channels; L-type; Cav1.2; AKAP79; AKAP18; Proline rich domain; Leucine Zipper motif
Background: PKA is confined to subcellular compartments by A-kinase anchoring proteins (AKAPs).
Results: A structure-based phage-directed evolution strategy has yielded modified PKA regulatory type II subunits with AKAP-selective binding properties.
Conclusion: Engineered RSelect proteins preferentially target particular AKAP-PKA interfaces
Significance: RSelect subunits are tools to distinguish and manipulate subpopulations of anchored PKA.
PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.
AKAP; Cell Biology; Peptide Arrays; Phage Display; Protein Kinase A (PKA); Compartmentalization; Structure-based Design
AIM: To examine trends in and correlates of liver disease and viral hepatitis in an human immunodeficiency virus (HIV)-infected cohort.
METHODS: The multi-site adult/adolescent spectrum of HIV-related diseases (ASD) followed 29 490 HIV-infected individuals receiving medical care in 11 U.S. metropolitan areas for an average of 2.4 years, and a total of 69 487 person-years, between 1998 and 2004. ASD collected data on the presentation, treatment, and outcomes of HIV, including liver disease, hepatitis screening, and hepatitis diagnoses.
RESULTS: Incident liver disease, chronic hepatitis B virus (HBV), and hepatitis C virus (HCV) were diagnosed in 0.9, 1.8, and 4.7 per 100 person-years. HBV and HCV screening increased from fewer than 20% to over 60% during this period of observation (P < 0.001). Deaths occurred in 57% of those diagnosed with liver disease relative to 15% overall (P < 0.001). Overall 10% of deaths occurred among individuals with a diagnosis of liver disease. Despite care guidelines promoting screening and vaccination for HBV and screening for HCV, screening and vaccination were not universally conducted or, if conducted, not documented.
CONCLUSION: Due to high rates of incident liver disease, viral hepatitis screening, vaccination, and treatment among HIV-infected individuals should be a priority.
Human immunodeficiency virus; Hepatitis B; Hepatitis C; Liver disease
Second messenger responses rely on where and when the enzymes that propagate these signals become active. Spatial and temporal organization of certain signaling enzymes is controlled in part by A-kinase anchoring proteins (AKAPs). This family of regulatory proteins was originally classified on the basis of their ability to compartmentalize the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A, or PKA). However, it is now recognized that AKAPs position G protein–coupled receptors, adenylyl cyclases, G proteins, and their effector proteins in relation to protein kinases and signal termination enzymes such as phosphodiesterases and protein phosphatases. This arrangement offers a simple and efficient means to limit the scope, duration, and directional flow of information to sites deep within the cell. This review focuses on the pros and cons of reagents that define the biological role of kinase anchoring inside cells and discusses recent advances in our understanding of anchored second messenger signaling in the cardiovascular and immune systems.
cell signaling; compartmentalization; cAMP; A-kinase anchoring proteins; protein phosphorylation
The mitogenic and second-messenger signals that promote cell proliferation often proceed through multienzyme complexes. The kinase-anchoring protein Gravin integrates cAMP and calcium/phospholipid signals at the plasma membrane by sequestering protein kinases A and C with G protein-coupled receptors. In this report we define a role for Gravin as a temporal organizer of phosphorylation-dependent protein-protein interactions during mitosis. Mass spectrometry, molecular, and cellular approaches show that CDK1/Cyclin B1 phosphorylates Gravin on threonine 766 to prime the recruitment of the polo-like kinase Plk1 at defined phases of mitosis. Fluorescent live-cell imaging reveals that cells depleted of Gravin exhibit mitotic defects that include protracted prometaphase and misalignment of chromosomes. Moreover, a Gravin T766A phosphosite mutant that is unable to interact with Plk1 negatively impacts cell proliferation. In situ detection of phospho-T766 Gravin in biopsy sections of human glioblastomas suggests that this phosphorylation event might identify malignant neoplasms.
Spatial and temporal organization of signal transduction is coordinated through the segregation of signaling enzymes in selected cellular compartments. This highly evolved regulatory mechanism ensures the activation of selected enzymes only in the vicinity of their target proteins. In this context, cAMP-responsive triggering of protein kinase A is modulated by a family of scaffold proteins referred to as A-kinase anchoring proteins. A-kinase anchoring proteins form the core of multiprotein complexes and enable simultaneous but segregated cAMP signaling events to occur in defined cellular compartments. In this review we will focus on the description of A-kinase anchoring protein function in the regulation of cardiac physiopathology.
AKAP; PKA; cAMP; cardiac disease
The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery.
Hsp20 (heat-shock protein of 20 kDa; HspB6) is a cardioprotective agent which combats a number of pathophysiological processes in the heart, including hypertrophy, apoptosis and ischaemia/reperfusion injury. The cardioprotective actions of Hsp20 require its phosphorylation by PKA (cAMP-dependent protein kinase) on Ser16. Although the extracellular stimuli that promote cAMP-responsive phosphorylation of Hsp20 are well known, less is understood about the molecular processes that regulate this modification. AKAPs (A-kinase-anchoring proteins) physically compartmentalize PKA to specific locations within a cell to both direct PKA phosphorylation toward selected substrates and to orchestrate downstream signalling events. In the present study we used PKA anchoring disruptor peptides to verify that an AKAP underpins the cardioprotective phosphorylation of Hsp20. Biochemical and immunofluorescence techniques identify the cytosolic protein AKAP-Lbc (AKAP13) as the anchoring protein responsible for directing PKA phosphorylation of Hsp20 on Ser16. Gene silencing and rescue experiments establish that AKAP-Lbc-mediated PKA phosphorylation of Hsp20 is crucial to the anti-apoptotic effects of the Hsp. Thus AKAP-Lbc may serve an ancillary cardioprotective role by favouring the association of PKA with Hsp20.
AKAP-Lbc; cAMP; cAMP-dependent protein kinase (PKA); cardioprotection; heat-shock protein of 20 kDa (Hsp20); phosphodiesterase (PDE)
AKAP150 knockout- and mutant knock-in alleles reveal an unexpected role of the adaptor in anchoring phosphatase 2B for efficient insulin secretion from pancreatic β-cells and thus glucose homeostasis.
Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.
A-kinase anchoring protein (AKAP); calcineurin (PP2B); cyclic-AMP-dependent protein kinase (PKA); glucoregulation; glucose-stimulated insulin secretion (GSIS)
A structured literature review was performed to detail the frequency and etiology of chronic liver disease (CLD) in Aboriginal North Americans. CLD affects Aboriginal North Americans disproportionately and is now one of the most common causes of death. Alcoholic liver disease is the leading etiology of CLD, but viral hepatitis, particularly hepatitis C, is an important and growing cause of CLD. High rates of autoimmune hepatitis and primary biliary cirrhosis (PBC) are reported in regions of coastal British Columbia and southeastern Alaska. Non-alcoholic liver disease is a common, but understudied, cause of CLD. Future research should monitor the incidence and etiology of CLD and should be geographically inclusive. In addition, more research is needed on the treatment of hepatitis C virus (HCV) infection and non-alcoholic fatty liver disease (NAFLD) in this population.
Hepatitis C virus; Hepatitis B virus; American Indian; Alaska Native; Chronic liver disease
Rac GTPases promote formation of membrane ruffles, yet how key effectors of this small GTPase operate in response to intracellular signals is not well established. In our recent report, “Anchored PKA recruitment of active Rac,” we identify a cortical actin cytoskeletal signaling complex containing an A-Kinase Anchoring Protein (AKAP) and the IQGAP2 isoform. We show that dynamic assembly of this complex requires the combined action of calcium and cAMP signals. Furthermore, phosphorylation of IQGAP2 by the AKAP220-anchored PKA enhances Rac binding and membrane ruffling. We also discuss our recent findings and provide additional evidence that phosphorylation of IQGAP2 brings IQGAP2 to membrane ruffles.
AKAP220; IQGAP2; PKA; GTPase; Rac; actin; membrane ruffles
L-type Ca2+ (CaV1.2) channels shape the cardiac action potential waveform and are essential for excitation-contraction coupling in heart. A gain-of-function G406R mutation in a cytoplasmic loop of CaV1.2 channels causes long QT syndrome 8 (LQT8), a disease also known as Timothy syndrome. However, the mechanisms by which this mutation enhances CaV1.2-LQT8 currents and generates lethal arrhythmias are unclear.
To test the hypothesis that the anchoring protein AKAP150 modulates CaV1.2-LQT8 channel gating in ventricular myocytes.
Methods and Results
Using a combination of molecular, imaging, and electrophysiological approaches, we discovered that CaV1.2-LQT8 channels are abnormally coupled to AKAP150. A pathophysiological consequence of forming this aberrant ion channel-anchoring protein complex is enhanced CaV1.2-LQT8 currents. This occurs through a mechanism whereby the anchoring protein functions like a subunit of CaV1.2-LQT8 channels that stabilizes the open conformation and augments the probability of coordinated openings of these channels. Ablation of AKAP150 restores normal gating in CaV1.2-LQT8 channels and protects the heart from arrhythmias.
We propose that AKAP150-dependent changes in CaV1.2-LQT8 channel gating may constitute a novel general mechanism for CaV1.2-driven arrhythmias.
CaV1.2 channels; EC coupling; calcium; arrhythmias
Phosphorylation is the predominant language of cell signaling. And, as with any common language, an abundance of dialects has evolved to convey complex information. We discuss here how biosensors are being used to decode this language, affording an unprecedented glimpse into spatio-temporal patterns of protein phosphorylation events within the cell.
A-Kinase Anchoring Proteins (AKAPs) coordinate cell-signaling events. AKAP79 brings together different combinations of enzyme binding partners to customize the regulation of effector proteins. In neurons muscarinic agonists mobilize an AKAP79-anchored pool of PKC that phosphorylates the KCNQ2 subunit of the M channel. This inhibits potassium permeability to enhance neuronal excitability. Using a dual fluorescent imaging/patch-clamp technique, we visualized AKAP79-anchored PKC phosphorylation of the kinase activity reporter CKAR concurrently with electrophysiological changes in KCNQ2 channels to show that AKAP79 synchronizes both signaling events to optimize the attenuation of M currents. AKAP79 also protects PKC from certain ATP competitive inhibitors. Related studies suggest that context dependent protein-protein interactions alter the susceptibility of another protein kinase, PDK1, to ATP analog inhibitors. This implies that intracellular binding partners not only couple individual molecular events in a cell signaling process but can also change the pharmacological profile of certain protein kinases.
To detail the clinical findings in a British family with molecularly characterised Wagner syndrome.
Only in the last year has the specific genetic defect in Wagner syndrome been identified, and the background literature of the molecular genetics is outlined. Clinical and laboratory findings in a second case of Wagner syndrome are included to highlight difficulties that can be encountered when identifying pathogenic mutations for disorders arising in complex genes.
Mutation screening was performed using PCR and RT‐PCR.
A heterozygous mutation was found converting the donor splice site of exon 8 of the chondroitin sulphate proteoglycan 2 (CSPG2). This is the same mutation that has been reported in the original Wagner pedigree. The main clinical features of Wagner syndrome are vitreous syneresis, thickening and incomplete separation of the posterior hyaloid membrane, chorioretinal changes accompanied by subnormal electroretinographic responses, an ectopic fovea and early‐onset cataract. A clinical feature present in this family, but previously undescribed, is anterior uveitis without formation of synechiae. Wagner syndrome has a progressive course, resulting in loss of vision even in the absence of retinal detachment.
On a background of considerable confusion regarding the distinction between Wagner syndrome and predominantly ocular Stickler syndrome, it is now apparent the that two conditions are both clinically and genetically distinct. This report summarises the clinical findings in Wagner syndrome and extends the phenotypic characteristics.
Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30 % of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser119 inside cells and that this mode of regulation may control its ability to affect cell growth.
kinase; peptide array; phosphoproteome; protein kinase A (PKA)