Neuronal microtubule-associated protein 2D is a dual a-kinase anchoring protein expressed in rat ovarian granulosa cells

A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype.     

A-kinase anchoring protein AKAP220 binds to glycogen synthase kinase-3beta (GSK-3beta ) and mediates protein kinase A-dependent inhibition of GSK-3beta

Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3beta binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3beta formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3beta, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3beta, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3beta bound to AKAP220 decreased more markedly than the total GSK-3beta activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3beta bound to AKAP220 more strongly than the total GSK-3beta activity. These results suggest that PKA and PP1 regulate the activity of GSK-3beta efficiently by forming a complex with AKAP220.     

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.     

The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c,regulates microtubule binding and stability

Phosphorylation of microtubule-associated protein 2 (MAP2) has a profound effect on microtubule stability and organization. In this work a consensus protein kinase A (PKA) phosphorylation site, T(220), of juvenile MAP2c is characterized. As confirmed by mass spectrometry, this site can be phosphorylated by PKA but shows less than average reactivity among the 3.5 +/- 0.5 phosphate residues incorporated into the protein. In contrast, T(220) is uniquely sensitive to dephosphorylation: three major Ser/Thr protein phosphatases, in the order of efficiency PP2B > PP2A(c) > PP1(c), remove this phosphate group first. MAP2c specifically dephosphorylated at this site binds and stabilizes microtubules stronger than either fully phosphorylated or nonphosphorylated MAP2c. Phosphorylation of this site also affects proteolytic sensitivity of MAP2c, which might represent a further level of control in this system. Thus, the phosphorylation state of T(220) may be a primary determinant of microtubule function.     

Critical role of cAMP-dependent protein kinase anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons

The cAMP-dependent protein kinase (PKA) regulates a wide array of cellular functions. In brain and heart PKA increases the activity of the L-type Ca2+ channel Cav1.2 in response to beta-adrenergic stimulation. Cav1.2 forms a complex with the beta2-adrenergic receptor, the trimeric GS protein, adenylyl cyclase, and PKA wherein highly localized signaling occurs [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Burette, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. PKA primarily phosphorylates Cav1.2 on serine 1928 of the central, pore-forming alpha11.2 subunit. Here we demonstrate that the A-kinase anchor protein 150 (AKAP150) is critical for PKA-mediated regulation of Cav1.2 in the brain. AKAP150 and MAP2B specifically co-immunoprecipitate with Cav1.2 from rat brain. Recombinant AKAP75, the bovine homologue to rat AKAP150, binds directly to three different sites of alpha11.2. MAP2B from rat brain also interacts with these same sites in pull-down assays. Gene disruption of AKAP150 in mice dramatically reduces co-immunoprecipitation of PKA with Cav1.2 and prevents phosphorylation of serine 1928 upon beta-adrenergic stimulation in vivo. These results demonstrate the physiological relevance of PKA anchoring by AKAPs in general and AKAP150 specifically in the regulation of Cav1.2 in vivo.     

Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.     

Enhancement of ganglioside GM3 synthesis in okadaic-acid-treated granulosa cells.

Okadaic acid is a potent inhibitor of type-2A (PP2A) and type-1 (PP1) protein phosphatases and has been proved to be a valuable tool for studies on the protein phosphorylation. We have investigated the effects of okadaic acid on rat granulosa cells in order to determine whether the regulation of ganglioside synthesis involves protein phosphorylation via inhibition of PP2A and PP1. Granulosa cells expressed luteinizing hormone (LH) receptors, measured as the binding of 125I-deglycosylated human chorionic gonadotropin (hCG) to intact cells, and synthesized the gangliosides NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer (GM3) and Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer (GM1), demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose, in response to follicle-stimulating hormone (FSH). When FSH-stimulated granulosa cells were treated with 10 nM okadaic acid for 15 h, down-regulation of LH receptors, dissociation of LH receptor-effector coupling and significant decreases of intracellular and extracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels were observed. The okadaic acid-induced desensitization to gonadotropin in granulosa cells was accompanied by increased ganglioside synthesis. The amount of 3H-labeled ganglioside GM3, the major ganglioside (about 95% of the total) synthesized by mature granulosa cells, was enhanced in okadaic acid-desensitized cells (to 215% of the control value) and in those desensitized by hCG (by 354%), forskolin (by 190%) and 12-O-tetradecanoylphorbol 13-acetate (by 143%). The results of this study suggest that an increase in the phosphorylation state of cells is accompanied by enhancement of ganglioside synthesis.     

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.     

GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling

A major determinant of neuronal morphology is the cytoskeleton. And one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of protein kinases and phosphatases present in neurons, including glycogen-synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3beta to analyze possible effects on microtubule stability and on the association of MAP2 with microtubules. We have found that GSK3beta phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Additionally, the formation of microtubule bundles, which is observed after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3beta wild type. Microtubule bundles were not observed in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3beta wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3beta wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein preparations. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.     

A role for AKAP (A kinase anchoring protein) scaffolding in the loss of a cyclic adenosine 3',5'-monophosphate inhibitory response in late pregnant rat myometrium

During pregnancy in the rat, there is a change in the ability of chlorophenylthio (CPT)-cAMP to inhibit myometrial phosphatidylinositide turnover. This is accompanied by a change in the association of proteins with a plasma membrane A kinase anchoring protein (AKAP). Both CPT-cAMP and isoproterenol inhibited oxytocin-stimulated phosphatidylinositide turnover on days 12 through 20 of gestation, whereas neither agent had an effect on day 21. Accompanying this change was a dramatic decrease in the concentration and activity of cAMP-dependent protein kinase [protein kinase A (PKA)] and an increase in the concentration of protein phosphatase 2B (PP2B) in plasma membranes from day 21 compared with day 19 pregnant rats. In contrast, both PKA and PP2B concentrations and activities increased in total myometrial homogenates. Both PKA and PP2B coimmunoprecipitated with an antibody against the 150-kDa AKAP found in rat myometrial plasma membranes. More PKA was associated with AKAP150 on day 19 than on day 21, while the reverse was true for PP2B. Disruption of PKA/AKAP association in day 19 pregnant rat myometrial cells with the specific interaction inhibitor peptide S-Ht31 resulted in the loss of the cAMP-inhibitory effect on phosphatidylinositide turnover. PP2B activity in myometrial homogenates dephosphorylated PLCbeta3, a PKA substrate targeted in the inhibition of Galphaq-stimulated phosphatidylinositide turnover. The dramatic loss of the cAMP-inhibitory effect on day 21 of pregnancy may alter the balance between uterine contraction and relaxation near parturition. The changes in the relative concentrations of PKA and PP2B associated with AKAP150 are consistent with a functional role for AKAP150 scaffolding in the alteration of cellular signaling.     

Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells

Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.     

PP2B/calcineurin-mediated desensitization of TRPV1 does not require AKAP150

Activation of protein kinases and phosphatases at the plasma membrane often initiates agonist-dependent signalling events. In sensory neurons, AKAP150 (A-kinase-anchoring protein 150) orientates PKA (protein kinase A), PKC (protein kinase C) and the Ca2+/calmodulin-dependent PP2B (protein phosphatase 2B, also known as calcineurin) towards membrane-associated substrates. Recent evidence indicates that AKAP150-anchored PKA and PKC phosphorylate and sensitize the TRPV1 (transient receptor potential subfamily V type 1 channel, also known as the capsaicin receptor). In the present study, we explore the hypothesis that an AKAP150-associated pool of PP2B catalyses the dephosphorylation and desensitization of TRPV1. Biochemical, electrophysiological and cell-based experiments indicate that PP2B associates with AKAP150 and TRPV1 in cultured TG (trigeminal ganglia) neurons. Gene silencing of AKAP150 reduces basal phosphorylation of TRPV1. However, functional studies in neurons isolated from AKAP150-/- mice indicate that the anchoring protein is not required for pharmacological desensitization of TRPV1. Behavioural analysis of AKAP150-/- mice further support this notion, demonstrating that agonist-stimulated desensitization of TRPV1 is sensitive to PP2B inhibition and does not rely on AKAP150. These findings allow us to conclude that pharmacological desensitization of TRPV1 by PP2B may involve additional regulatory components.     

RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.     

Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta

Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin alpha 2 beta 1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, alpha 2 beta 1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3 beta (GSK3 beta) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the alpha 2 cytoplasmic domain and by a function-blocking anti-alpha 2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin alpha 2 beta 1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the alpha 2 beta 1-induced activation of PP2A.     

Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as K(153)GVLF(157). PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PP1 from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis.     

Cellular mechanisms and modulation of activin A- and transforming growth factor beta-mediated differentiation in cultured hen granulosa cells

Undifferentiated granulosa cells from prehierarchal (6- to 8-mm-diameter) hen follicles express very low to undetectable levels of LH receptor (LH-R) mRNA, P450 cholesterol side chain cleavage (P450scc) enzyme activity, and steroidogenic acute regulatory (StAR) protein, and produce negligible progesterone, in vitro, following an acute (3-h) challenge with either FSH or LH. It has previously been established that culturing such cells with FSH for 18-20 h induces LH-R, P450scc, and StAR expression, which enables the initiation of progesterone production. The present studies were conducted to characterize the ability of activin and transforming growth factor (TGF) beta, both alone and in combination with FSH, to promote hen granulosa cell differentiation, in vitro. A 20-h culture of prehierarchal follicle granulosa cells with activin A or transforming growth factor beta (TGFbeta)1 increased LH-R mRNA levels compared with control cultured cells. Activin A and TGFbeta1 also promoted FSH-receptor (FSH-R) mRNA expression when combined with FSH treatment. Neither activin A nor TGFbeta1 alone stimulated progesterone production after 20 h culture. However, preculture with either factor for 20 h (to induce gonadotropin receptor mRNA expression) followed by a 3-h challenge with FSH or LH potentiated StAR expression and progesterone production compared with cells challenged with gonadotropin in the absence of activin A or TGFbeta1 preculture. Significantly, activation of the mitogen-activated protein (MAP) kinase pathway with transforming growth factor alpha (TGFalpha) (monitored by Erk phosphorylation) blocked TGFbeta1-induced LH-R expression, and this effect was associated with the inhibition of Smad2 phosphorylation. We conclude that a primary differentiation-inducing action of activin A and TGFbeta1 on hen granulosa cells from prehierarchal follicles is directed toward LH-R expression. Enhanced LH-R levels subsequently sensitize granulosa cells to LH, which in turn promotes StAR plus P450scc expression and subsequently an increase in P4 production. Significantly, the finding that TGFbeta signaling is negatively regulated by MAP kinase signaling is proposed to represent a mechanism that prevents premature differentiation of granulosa cells.     

The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca(2+) channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming alpha(1) subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with (32)P-labeled RIIbeta revealed a prominent AKAP with an M(r) of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the alpha(1) subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca(2+) channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca(2+) channels. Accordingly, MAP2B docked at class C Ca(2+) channels may be important for recruiting PKA to postsynaptic sites.     

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.