Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220

The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610–623) preferentially interacts with RII, whereas site 2 (residues 1633–1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β.     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

Anchored phosphatases modulate 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.     

Cytokine-induced activation of mixed lineage kinase 3 requires TRAF2 and TRAF6

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple mitogen-activated protein kinase (MAPK) pathways in response to growth factors, stresses and the pro-inflammatory cytokine, tumor necrosis factor (TNF). MLK3 is required for optimal activation of stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling by TNF, however, the mechanism by which MLK3 is recruited and activated by the TNF receptor remains poorly understood. Here we report that both TNF and interleukin-1β (IL-1β) stimulation rapidly activate MLK3 kinase activity. We observed that TNF stimulates an interaction between MLK3 and TNF receptor associated factor (TRAF) 2 and IL-1β stimulates an interaction between MLK3 and TRAF6. RNA interference (RNAi) of traf2 or traf6 dramatically impairs MLK3 activation by TNF indicating that TRAF2 and TRAF6 are critically required for MLK3 activation. We show that TNF also stimulates ubiquitination of MLK3 and MLK3 can be conjugated with lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitin chains. Our results suggest that K48-linked ubiquitination directs MLK3 for proteosomal degradation while K63-linked ubiquitination is important for MLK3 kinase activity. These results reveal a novel mechanism for MLK3 activation by the pro-inflammatory cytokines TNF and IL-1β.     

Coordination of Protein Phosphorylation and Dephosphorylation in Synaptic Plasticity

A central theme in nervous system function is equilibrium: synaptic strengths wax and wane, neuronal firing rates adjust up and down, and neural circuits balance excitation with inhibition. This push/pull regulatory theme carries through to the molecular level at excitatory synapses, where protein function is controlled through phosphorylation and dephosphorylation by kinases and phosphatases. However, these opposing enzymatic activities are only part of the equation as scaffolding interactions and assembly of multi-protein complexes are further required for efficient, localized synaptic signaling. This review will focus on coordination of postsynaptic serine/threonine kinase and phosphatase signaling by scaffold proteins during synaptic plasticity.     

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.     

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser⁷⁸⁹. Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.     

LPA-producing enzyme PA-PLA₁α regulates hair follicle development by modulating EGFR signalling

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.     

The A-kinase Anchoring Protein GSKIP Regulates GSK3β Activity and Controls Palatal Shelf Fusion in Mice

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5, GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.