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.     

Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.     

Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.

Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.     

Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79.

Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.     

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.     

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.     

Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.     

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.     

Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP)

Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.     

cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta.

In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.     

A-kinase anchor proteins as potential regulators of protein kinase A function in oocytes

In the mammalian oocyte, the cAMP-dependent protein kinase (PKA) has critical functions in the maintenance of meiotic arrest and oocyte maturation. Because PKA is spatially regulated, its localization was examined in developing oocytes. Both regulatory subunits (RI and RII) and the catalytic subunit (C) of PKA were found in oocytes and metaphase II-arrested eggs. In the oocyte, RI and C were predominantly localized in the cortical region, while RII showed a punctate distribution within the cytoplasm. After maturation to metaphase II, RI remained in the cortex and was also localized to the meiotic spindle, while RII was found adjacent to the spindle. C was diffuse within the cytoplasm of the egg but was enriched in the cytoplasm surrounding the metaphase spindle, much like RII. The polarized localization and redistribution of RI, RII, and C suggested that PKA might be tethered by A-kinase anchor proteins (AKAPs), proteins that tether PKA close to its physiological substrates. An AKAP, AKAP140, was identified that was developmentally regulated and phosphorylated in oocytes and eggs. AKAP140 was shown to be a dual-specific AKAP, having the ability to bind both RI and RII. By compartmentalizing PKA, AKAP140 and/or other AKAPs could spatially regulate PKA activity during oocyte development.     

Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA

The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.     

The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis

Protein kinase A (PKA) and the nuclear A-kinase-anchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95-PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RIIalpha regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306-carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation.

Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.