AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.     

Unconventional myosin VIIA is a novel A-kinase-anchoring protein

To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.     

Synergistic effect of histone H1 and nucleolin on chromatin condensation in mitosis: role of a phosphorylated heteromer

Repeated motifs, rich in basic residues, are characteristic of both the N-terminal domain of the nucleolus-specific protein, nucleolin, and the second half of the C-terminal domain of histone H1. These repeats are also the target for phosphorylation by the mitosis-specific p34cdc2 kinase. We have previously shown that synthetic peptides [(KTPKKAKKP)2 for histone H1 and (ATPAKKAA)2 for nucleolin] corresponding to these two repeated motifs are able to act in synergy to induce DNA hypercondensation (Erard et al., 1990). In order to determine the molecular basis of this synergistic interaction, we have studied the condensation of the homopolymer poly(dA).poly(dT) in the presence of the two synthetic peptides. Circular dichroism has been used to monitor the psi (+)-type condensation and has revealed that phosphorylation enhances the synergistic effect of the two peptides. Analysis of different combinations of the two peptides suggests that there is a direct interaction between them which is stabilized by phosphorylation. Furthermore, there is a striking correlation between the degree of homopolymer condensation and the stability of the heteromeric complex. Phosphorylation takes place on the threonine residues on the repeat motifs within a region which is likely to adopt a beta-turn structure. Circular dichroism and infrared spectroscopy provide evidence that phosphorylation stabilizes the beta-turn structure of both peptides, and computer modeling shows that this may be due to steric hindrance imposed by the phosphate group. We suggest that phosphorylated nucleolin and histone H1 interact through their homologous domain structured in beta-spirals in order to condense certain forms of DNA during mitosis.     

AMY-1 interacts with S-AKAP84 and AKAP95 in the cytoplasm and the nucleus,respectively, and inhibits cAMP-dependent protein kinase activity by preventing binding of its catalytic subunit to A-kinase-anchoring protein (AKAP) complex

We have reported that a novel c-Myc-binding protein, AMY-1, binds to cAMP-dependent protein kinase-anchoring protein 149 (AKAP149) and its splicing variant, AKAP84 and is localized in the mitochondria in a complex with RII, a regulatory subunit of cAMP-dependent protein kinase (PKA) (Furusawa, M., Ohnishi, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2001) J. Biol. Chem. 276, 36647-36651). In this study, we further found that AMY-1 competitively bound to either AKAP95 or AKAP84 in the nucleus and the cytoplasm, respectively, in a concentration-dependent manner of either AKAP. Like AKAP84, AMY-1 was found to bind to the RII-binding region of AKAP95 in vivo and in vitro and to make a ternary complex with RII. It was also found that the formation of the complex of AMY-1 with AKAP84/95 and RII prevented a catalytic subunit from binding to this AKAP complex, leading to suppression of PKA activity. These findings suggest that AMY-1 is an important modulator of PKA.     

Interaction between fidgetin and protein kinase A-anchoring protein AKAP95 is critical for palatogenesis in the mouse

The gene defective in fidget mice encodes fidgetin, a member of the AAA (ATPases associated with diverse cellular activities) family of ATPases. Using a yeast two-hybrid screen, we identified cAMP-dependent protein kinase A anchoring protein 95 kDa (AKAP95) as a potential fidgetin-binding protein. Epitope-tagged fidgetin co-localized with endogenous AKAP95 in the nuclear matrix, and the physical interaction between fidgetin and AKAP95 was further confirmed by reciprocal immunoprecipitation. To evaluate the biological significance of the fidgetin-AKAP95 binding, we created AKAP95 mutant mice through a gene trap strategy. Akap95 mutant mice are surprisingly viable with no overt phenotype. However, a significant number of mice carrying both Akap95 and fidget mutations die soon after birth due to cleft palate, consistent with the overlapping expression of AKAP95 and fidgetin in the branchial arches during mouse embryogenesis. These results expand the spectrum of the pleiotropic phenotypes of fidget mice and provide new leads on the in vivo function of AKAP95.     

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.     

Distinct but overlapping domains of AKAP95 are implicated in chromosome condensation and condensin targeting

A-kinase (or PKA)-anchoring protein AKAP95 is a zinc-finger protein implicated in mitotic chromosome condensation by acting as a targeting molecule for the condensin complex. We have identified determinants of chromatin-binding, condensin-targeting and chromosome-condensation activities of AKAP95. Binding of AKAP95 to chromatin is conferred by residues 387–450 and requires zinc finger ZF1. Residues 525–569 are essential for condensation of AKAP95-free chromatin and condensin recruitment to chromosomes. Mutation of either zinc finger of AKAP95 abolishes condensation. However, ZF1 is dispensable for condensin targeting, whereas the C-terminal ZF2 is required. AKAP95 interacts with Xenopus XCAP-H condensin subunit in vitro and in vivo but not with the human hCAP-D2 subunit. The data illustrate the involvement of overlapping, but distinct, domains of AKAP95 for condensin recruitment and chromosome condensation and argue for a key role of ZF1 in chromosome condensation and ZF2 in condensin targeting. Moreover, condensin recruitment to chromatin is not sufficient to promote condensation.     

Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication

Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.     

A kinase-anchoring protein (AKAP)95 recruits human chromosome-associated protein (hCAP)-D2/Eg7 for chromosome condensation in mitotic extract

Association of the condensin multiprotein complex with chromatin is required for chromosome condensation at mitosis. What regulates condensin targeting to chromatin is largely unknown. We previously showed that the nuclear A kinase-anchoring protein, AKAP95, is implicated in chromosome condensation. We demonstrate here that AKAP95 acts as a targeting protein for human chromosome-associated protein (hCAP)-D2/Eg7, a component of the human condensin complex, to chromosomes. In HeLa cell mitotic extract, AKAP95 redistributes from the nuclear matrix to chromatin. When association of AKAP95 with chromatin is prevented, the chromatin does not condense. Condensation is rescued by a recombinant AKAP95 peptide containing the 306 COOH-terminal amino acids of AKAP95. Recombinant AKAP95 binds chromatin and elicits recruitment of Eg7 to chromosomes in a concentration-dependent manner. Amount of Eg7 recruited correlates with extent of chromosome condensation: resolution into distinct chromosomes is obtained only when near-endogenous levels of Eg7 are recruited. Eg7 and AKAP95 immunofluorescently colocalize to the central region of methanol-fixed metaphase chromosomes. GST pull-down data also suggest that AKAP95 recruits several condensin subunits. The results implicate AKAP95 as a receptor that assists condensin targeting to chromosomes.     

Acinus-provoked protein kinase C delta isoform activation is essential for apoptotic chromatin condensation

Histone H2B phosphorylation tightly correlates with chromatin condensation during apoptosis. The caspase-cleaved acinus (apoptotic chromatin condensation inducer in the nucleus) provokes chromatin condensation in the nucleus, but the molecular mechanism accounting for this effect remains elusive. Here, we report that the active acinus p17 fragment initiates H2B phosphorylation and chromatin condensation by activating protein kinase C delta isoform (PKC-delta). We show that p17 binds to both Mst1 and PKC-delta, which is upregulated by apoptotic stimuli, enhancing their kinase activities. Acinus mutant susceptible to degradation elicits stronger chromatin condensation and higher H2B phosphorylation than wild-type acinus. Dominant-negative PKC-delta but not Mst1 robustly blocks acinus-initiated H2B phosphorylation. Surprisingly, depletion of Mst1 triggers caspase-3 activation, provoking H2B phosphorylation through activating PKC-delta. Further, acinus-elicited H2B phosphorylation and chromatin condensation are abrogated in PKC-delta-deficient mouse embryonic fibroblast cells and siRNA-knocked down PC12 cells. Thus, PKC-delta but not Mst1 acts as a physiological downstream kinase of acinus in promoting H2B phosphorylation and chromatin condensation.     

Pericentric chromatin is organized into an intramolecular loop in mitosis

BACKGROUND: Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown. RESULTS: We provide evidence that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA adopts an intramolecular loop, whereas sister-chromatid arms are paired intermolecularly. Visualization of cohesin subunits by fluorescence microscopy revealed a cylindrical structure that encircles the central spindle and spans the distance between sister kinetochores. Kinetochore assembly at the apex of the loop initiates intrastrand loop formation that extends approximately 25 kb (12.5 kb on either side of the centromere). Two centromere loops (one from each sister chromatid) are stretched between the ends of sister-kinetochore microtubules along the spindle axis. At the base of the loop there is a transition to intermolecular sister-chromatid pairing. CONCLUSIONS: The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.     

Cysteine-protease involved in male chromatin remodeling after fertilization co-localizes with alpha-tubulin at mitosis

We postulated an essential role for a cysteine-protease in sea urchins sperm histones degradation which follows fertilization. We now report the purification of this enzyme, the determination of its N-terminal amino acid sequence and the localization of the protein with antibodies generated against this amino-terminal peptide. The immunofluorescence data confirmed the presence of this enzyme in the nucleus of unfertilized eggs. After fertilization labeling is observed both in female and male pronuclei suggesting a rapid recruitment of the enzyme to the male pronuclei. Interestingly, we have found that this cysteine-protease persists in the nucleus of the zygotes during S phase of the cell cycle and co-localizes with alpha-tubulin that organizes the mitotic spindle during the initial embryonic cell division.     

The intermediate filament protein, synemin, is an AKAP in the heart

Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes.