AKAP350 modulates microtubule dynamics

AKAP350 is a multiply spliced type II protein kinase A-anchoring protein that localizes to the centrosomes in most cells and the Golgi apparatus in epithelial cells. Multiple studies suggest that AKAP350 is involved in microtubule nucleation at the centrosome. Our previous studies demonstrated that AKAP350 was necessary for the maintenance of Golgi apparatus integrity. These data suggested that AKAP350 might be necessary for normal cytoskeletal interactions with the Golgi. To examine the relationship of AKAP350 with the microtubule cytoskeleton, we analyzed the effect of the depletion of AKAP350 on microtubule regrowth after nocodazole treatment in HeLa cells. The decrease in AKAP350 expression with short interfering RNA induced a delay in microtubule elongation with no effect on microtubule aster formation. In contrast, overexpression of the centrosomal targeting domain of AKAP350 elicited alterations in aster formation, but did not affect microtubule elongation. RNA interference for AKAP350 also induced an increase in cdc42 activity during microtubule regrowth. Our data suggest that AKAP350 has a role in the remodeling of the microtubule cytoskeleton.     

AKAP350 at the Golgi apparatus. I. Identification of a distinct Golgi apparatus targeting motif in AKAP350

The protein kinase A-anchoring proteins (AKAPs) are defined by their ability to scaffold protein kinase A to specific subcellular compartments. Each of the AKAP family members utilizes unique targeting domains specific for a particular subcellular compartment. AKAP350 is a multiply spliced AKAP family member localized to the centrosome and the Golgi apparatus. Three splicing events in the carboxyl terminus of AKAP350 generate the AKAP350A, AKAP350B, and AKAP350C proteins. A monoclonal antibody recognizing all three splice variants as well as a polyclonal antibody specific for AKAP350A demonstrated both centrosomal and Golgi apparatus staining in paraformaldehyde-fixed HCA-7 cells. Golgi apparatus-associated AKAP350A staining was dispersed following brefeldin A treatment. Using GFP chimeric constructs of the carboxyl-terminal regions of AKAP350A, a Golgi apparatus targeting domain was identified between amino acids 3259 and 3307 of AKAP350A. This domain was functionally distinguishable from the recently described centrosomal targeting domain (PACT domain, amino acids 3308-3324) located adjacent to the Golgi targeting domain. These data definitively establish the specific association of AKAP350A with the Golgi apparatus in HCA-7 cells.     

AKAP350 Is involved in the development of apical "canalicular" structures in hepatic cells HepG2

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.     

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.     

AKAP350 at the Golgi apparatus. II. Association of AKAP350 with a novel chloride intracellular channel (CLIC) family member

AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit parietal cell library with a 3.2-kb segment of AKAP350 (nucleotides 3611-6813). This screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1). CLIC1 belongs to a family of proteins, all of which contain a high degree of homology in their carboxyl termini. All CLIC family members were able to bind a 133-amino acid domain within AKAP350 through the last 120 amino acids in the conserved CLIC carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC proteins recognized at least five CLIC species including a novel 46-kDa CLIC protein. We isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A splice variant of CLIC5, the predicted molecular mass of CLIC5B corresponds to the molecular mass of the 46-kDa CLIC immunoreactive protein in HCA-7 cells. Antibodies against CLIC5B colocalized with AKAP350 at the Golgi apparatus with minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi elements was lost following brefeldin A treatment. Furthermore, GFP-CLIC5B-(178-410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350 associates with CLIC proteins and specifically that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells.     

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.     

CLIC4 is enriched at cell-cell junctions and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells

CLIC4 is a member of the chloride intracellular channel (CLIC) protein family whose principal cellular functions are poorly understood. Recently, we demonstrated that several CLIC proteins, including CLIC4, interact with AKAP350. AKAP350 is concentrated at the Golgi apparatus, centrosome, and midbody and acts as a scaffolding protein for several protein kinases and phosphatases. In this report, we show that endogenous CLIC4 and AKAP350 colocalize at the centrosome and midbody of cultured cells by immunofluorescence microscopy. Unlike AKAP350, CLIC4 is not enriched in the Golgi apparatus but is enriched in mitochondria, actin-based structures at the cell cortex, and the nuclear matrix, indicating that CLIC4-AKAP350 interactions are regulated at specific subcellular sites in vivo. In addition to the centrosome and midbody, CLIC4 colocalizes with AKAP350 and the tight junction protein ZO-1 in the apical region of polarized epithelial cells, suggesting that CLIC4 may play a role in maintaining apical-basolateral membrane polarity during mitosis and cytokinesis. Biochemical studies show that CLIC4 behaves mainly as a soluble cytosolic protein and can associate with proteins of the microtubule cytoskeleton. The localization of CLIC4 to the cortical actin cytoskeleton and its association with AKAP350 at the centrosome and midbody suggests that CLIC4 may be important for regulating cytoskeletal organization during the cell cycle. These findings lead to the conclusion that CLIC4 and possibly other CLIC proteins have alternate cellular functions that are distinct from their proposed roles as chloride channels.     

A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors

Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991–1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward.     

Conservation and function of a bovine sperm A-kinase anchor protein homologous to mouse AKAP82.

Protein kinase A regulates sperm motility through the cAMP-dependent phosphorylation of proteins. One mechanism to direct the activity of the kinase is to localize it near its protein substrates through the use of anchoring proteins. A-Kinase anchoring proteins (AKAPs) act by binding the type II regulatory subunit of protein kinase A and tethering it to a cellular organelle or cytoskeletal element. We showed previously that mAKAP82, the major protein of the fibrous sheath of the mouse sperm flagellum, is an AKAP. The available evidence indicates that protein kinase A is compartmentalized to the fibrous sheath by binding mAKAP82. To characterize AKAP82 in bovine sperm, a testicular cDNA library was constructed and used to isolate a clone encoding bAKAP82, the bovine homologue. Sequence analysis showed that the primary structure of bAKAP82 was highly conserved. In particular, the amino acid sequence corresponding to the region of mAKAP82 responsible for binding the regulatory subunit of protein kinase A was identical in the bull. Bovine AKAP82 was present in both epididymal and ejaculated sperm and was localized to the entire principal piece of the flagellum, the region in which the fibrous sheath is located. Finally, bAKAP82 bound the regulatory subunit of protein kinase A. These data support the idea that bAKAP82 functions as an anchoring protein for the subcellular localization of protein kinase A in the flagellum.     

Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.     

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.     

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.     

Mapping the protein phosphatase-2B anchoring site on AKAP79. Binding and inhibition of phosphatase activity are mediated by residues 315-360

Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinase anchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes with N-methyl-d-aspartic acid or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315-360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315-360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330-357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity.     

The scaffolding protein CG-NAP/AKAP450 is a critical integrating component of the LFA-1-induced signaling complex in migratory T cells

T cell migration represents a complex highly coordinated process involving participation of surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. Members of the A-kinase anchoring protein (AKAP) family of giant scaffolding proteins can assemble and compartmentalize multiple signaling and structural molecules thereby providing a platform for their targeted positioning and efficient interactions. We characterize here the expression, intracellular distribution, and functional role of the scaffolding protein CG-NAP (centrosome and Golgi localized protein kinase N-associated protein)/AKAP450 in the process of active T cell motility induced via LFA-1 integrins. This protein is predominantly localized at the centrosome and Golgi complex. T cell locomotion triggered by LFA-1 ligation induces redistribution of CG-NAP/AKAP450 along microtubules in trailing cell extensions. Using an original in situ immunoprecipitation approach, we show that CG-NAP/AKAP450 is physically associated with LFA-1 in the multimolecular signaling complex also including tubulin and the protein kinase C beta and delta isoenzymes. CG-NAP/AKAP450 recruitment to this complex was specific for the T cells migrating on LFA-1 ligands, but not on the beta(1) integrin ligand fibronectin. Using the GFP-tagged C-terminal CG-NAP/AKAP450 construct, we demonstrate that expression of the intact CG-NAP/AKAP450 and its recruitment to the LFA-1-associated multimolecular complex is critically important for polarization and migration of T cells induced by this integrin.     

Identification and characterization of RHOA-interacting proteins in bovine spermatozoa

In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro.     

Identification and mapping of protein kinase A binding sites in the costameric protein myospryn

Recently we identified a novel target gene of MEF2A named myospryn that encodes a large, muscle-specific, costamere-restricted alpha-actinin binding protein. Myospryn belongs to the tripartite motif (TRIM) superfamily of proteins and was independently identified as a dysbindin-interacting protein. Dysbindin is associated with alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex (DGC) in muscle. Apart from these initial findings little else is known regarding the potential function of myospryn in striated muscle. Here we reveal that myospryn is an anchoring protein for protein kinase A (PKA) (or AKAP) whose closest homolog is AKAP12, also known as gravin/AKAP250/SSeCKS. We demonstrate that myospryn co-localizes with RII alpha, a type II regulatory subunit of PKA, at the peripheral Z-disc/costameric region in striated muscle. Myospryn interacts with RII alpha and this scaffolding function has been evolutionarily conserved as the zebrafish ortholog also interacts with PKA. Moreover, myospryn serves as a substrate for PKA. These findings point to localized PKA signaling at the muscle costamere.     

Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis

The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.     

A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines

The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.     

A High-Affinity Binding Protein for the Regulatory Subunit of cAMP-Dependent Protein Kinase II in the Centrosome of Human Cells

In the human lymphoblastic cell line KE 37, Northern blot analysis with cDNA probes for human regulatory subunits RIIα and RIIβ of the cAMP-dependent protein kinase (A-kinase) type II and immunoblotting or immunoprecipitation studies with several antibodies directed against RIIα and RIIβ show that these two isoforms are expressed. The major isoform α is mostly cytosolic, whereas the β isoform appears concentrated in the Golgi-centrosomal area, as judged by immunofluorescence and cell fractionation. Using a ³²P-labeled RII overlay on Western blots, a 350-kDa RII-binding protein (AKAP 350) was specifically identified in centrosomes isolated from this cell line, whereas a Golgi fraction has previously been demonstrated to contain an 85-kDa RII-binding protein (AKAP 85). AKAP 350 is highly insoluble and can partially be extracted from centrosomes as a complex of AKAP 350 and RII subunit. AKAP 350 was identified as a specific centrosomal protein previously demonstrated in the pericentriolar material. The potential significance of a specific subcellular distribution for different RII-binding proteins in nonneuronal cells is discussed.