Low Expression of miR-424-3p is Highly Correlated with Clinical Failure in Prostate Cancer.

Prostate cancer (PC) is a highly heterogenous disease and one of the leading causes of mortality in developed countries. Recently, studies have shown that expression of immune checkpoint proteins are directly or indirectly repressed by microRNAs (miRs) in many types of cancers. The great advantages of using miRs based therapy is the capacity of these short transcripts to target multiple molecules for the same- or different pathways with synergistic immune inhibition effects. miR-424 has previously been described as a biomarker of poor prognosis in different types of cancers. miR-424 is also found to target both the CTLA-4/CD80- and PD-1/PD-L1 axis. In the present study, the clinical significance of miR-424-3p expression in PC tissue was evaluated. Naïve radical prostatectomy specimens from 535 patients was used for tissue microarray construction. In situ hybridization was used to evaluate the expression of miR-424-3p and immunohistochemistry was used for CTLA-4 protein detection. In univariate- and multivariate analyses, low expression of miR-424-3p was significant associated with clinical failure-free survival, (p = 0.004) and p = 0.018 (HR:0.44, CI95% 0.22-0.87). Low expression of miR-424-3p also associated strongly with aggressive phenotype of PC. This highlight the importance of miR-424-3p as potential target for therapeutic treatment in prostate cancer.

Prognostic biomarkers for immunotherapy with ipilimumab in metastatic melanoma.

New therapies, including the anti-cytotoxic T lymphocyte antigen (CTLA)-4 antibody, ipilimumab, is approved for metastatic melanoma. Prognostic biomarkers need to be identified, because the treatment has serious side effects. Serum samples were obtained before and during treatment from 56 patients with metastatic or unresectable malignant melanoma, receiving treatment with ipilimumab in a national Phase IV study (NCT0268196). Expression of a panel of 17 inflammatory-related markers reflecting different pathways including extracellular matrix remodeling and fibrosis, vascular inflammation and monocyte/macrophage activation were measured at baseline and the second and/or third course of treatment with ipilimumab. Six candidate proteins [endostatin, osteoprotegerin (OPG), C-reactive protein (CRP), pulmonary and activation-regulated chemokine (PARC), growth differentiation factor 15 (GDF15) and galectin-3 binding-protein (Gal3BP)] were persistently higher in non-survivors. In particular, high Gal3BP and endostatin levels were also independently associated with poor 2-year survival after adjusting for lactate dehydrogenase, M-stage and number of organs affected. A 1 standard deviation increase in endostatin gave 1·74 times [95% confidence interval (CI) = 1·10-2·78, P = 0·019] and for Gal3BP 1·52 times (95% CI = 1·01-2·29, P = 0·047) higher risk of death in the adjusted model. Endostatin and Gal3BP may represent prognostic biomarkers for patients on ipilimumab treatment in metastatic melanoma and should be further evaluated. Owing to the non-placebo design, we could only relate our findings to prognosis during ipilimumab treatment.

Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

Targeting of type I protein kinase A to lipid rafts is required for platelet inhibition by the 3',5'-cyclic adenosine monophosphate-signaling pathway.

BACKGROUND: Platelet adhesion to von Willebrand factor (VWF) is modulated by 3',5'-cyclic adenosine monophosphate (cAMP) signaling through protein kinase A (PKA)-mediated phosphorylation of glycoprotein (GP)Ibß. A-kinase anchoring proteins (AKAPs) are proposed to control the localization and substrate specificity of individual PKA isoforms. However, the role of PKA isoforms in regulating the phosphorylation of GPIbß and platelet response to VWF is unknown. OBJECTIVES: We wished to determine the role of PKA isoforms in the phosphorylation of GPIbß and platelet activation by VWF as a model for exploring the selective partitioning of cAMP signaling in platelets. RESULTS: The two isoforms of PKA in platelets, type I (PKA-I) and type II (PKA-II), were differentially localized, with a small pool of PKA-I found in lipid rafts. Using a combination of Far Western blotting, immunoprecipitation, proximity ligation assay and cAMP pull-down we identified moesin as an AKAP that potentially localizes PKA-I to rafts. Introduction of cell-permeable anchoring disruptor peptide, RI anchoring disruptor (RIAD-Arg11 ), to block PKA-I/AKAP interactions, uncoupled PKA-RI from moesin, displaced PKA-RI from rafts and reduced kinase activity in rafts. Examination of GPIbß demonstrated that it was phosphorylated in response to low concentrations of PGI2 in a PKA-dependent manner and occurred primarily in lipid raft fractions. RIAD-Arg11 caused a significant reduction in raft-localized phosphoGPIbß and diminished the ability of PGI2 to regulate VWF-mediated aggregation and thrombus formation in vitro. CONCLUSION: We propose that PKA-I-specific AKAPs in platelets, including moesin, organize a selective localization of PKA-I required for phosphorylation of GPIbß and contribute to inhibition of platelet VWF interactions.

Compartmentalization of cAMP signaling in adipogenesis, lipogenesis, and lipolysis.

Energy storage and release at times of food excess or fasting are carefully coordinated processes that depend on the appropriate differentiation of mesenchymal stem cells into mature adipocytes (adipogenesis) forming white adipose tissue (WAT) and on regulatory signals for storage (lipogenesis) or mobilization (lipolysis) of triacylglycerides (TAGs) from lipid droplets. It is widely recognized that cAMP signaling via protein kinase A (PKA) is important both in adipogenesis and for hormonal control and lipolysis in WAT. A kinase anchoring proteins (AKAPs) target PKA to distinct subcellular compartments in close proximity to its specific substrates thereby providing spatial and temporal specificity in the mediation of biological effects controlled by the cAMP-PKA pathway. This review will provide an updated overview of some of the sites of regulation by cAMP in adipogenesis and lipolysis and the involvement of AKAPs and highlighting, as a recent example, the AKAP Optical Atrophy 1 (OPA1) and its role in the phosphorylation of Perilipin to induce lipolysis.

Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets.

BACKGROUND: Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high-throughput methodology for the quantitative analysis of platelet signaling events is still lacking. OBJECTIVE: To develop a high-throughput assay for the analysis of platelet signaling events in whole blood. METHODS AND RESULTS: We developed a fluorescent barcoding protocol to facilitate multiplexing and enable large-scale signaling profiling in platelets in whole blood. The methodology allowed simultaneous staining and acquisition of 24-96 samples in a single analysis tube with a standard flow cytometer. This approach significantly reduced experimental numbers, data acquisition time, and antibody consumption, while providing automated statistically rich quantitative data on signaling events. Using vasodilator-stimulated phosphoprotein (VASP), an established marker of platelet inhibition and antiplatelet drug therapy, we demonstrated that the assay could detect subtle changes in phosphoVASP-Ser157/239 in response to cAMP-elevating agents of varying potency and known modulators of the cAMP signaling cascade. The assay could be used with washed platelets or whole blood, analyzed immediately or frozen, without any significant change in assay performance. To demonstrate the usefulness of the assay as a drug discovery platform, we examined a prostaglandin screening library. Our screen of 70 prostaglandin derivatives revealed three previously uncharacterized lipids that stimulated phosphorylation of VASP-Ser157. Follow-up analyses demonstrated that these agents elevated intraplatelet cAMP and inhibited collagen-induced platelet aggregation. CONCLUSIONS: This novel method enables rapid, large-scale quantitative signaling profiling and compound screening in human platelets present in whole blood.

Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia.

The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms ß and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication.

Review: Spatiotemporal dynamics of hCG/cAMP signaling and regulation of placental function.

The pregnancy hormone human chorionic gonadotropin (hCG) is essential to sustain early pregnancy and involved in regulation of progesterone production, decidualization, and cytotrophoblast differentiation. It binds to and activates the G-protein coupled luteinizing hormone/hCG-receptor, activating the cAMP/protein kinase A (PKA) pathway which results in the phosphorylation of specific intracellular target proteins. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by phosphodiesterases and by discrete spatial and temporal activation of PKA in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins. Here we discuss spatiotemporal regulation of PKA signaling in response to hCG controlling placental function.

Effects of type I protein kinase A modulation on the T cell distal pole complex.

The distal pole complex (DPC) assembles signalling proteins at the T cell pole opposite the immunological synapse (IS) and is thought to facilitate T cell activation by sequestering negative regulatory molecules away from the T cell receptor-proximal signalling machinery. Here, we report the translocation of type I protein kinase A (PKA) to the DPC in a fraction of T cells following activation and the localization of type I PKA with known components of the DPC. We propose that sequestration of type I PKA and concomitant loss of cAMP-mediated negative regulation at the IS may be necessary to allow full T cell activation. Moreover, composition of the DPC appears to be modulated by type I PKA activity, as the antagonist Rp-8-Br-cAMPS inhibited translocation of type I PKA and other DPC proteins.

Combined Env- and Gag-specific T cell responses in relation to programmed death-1 receptor and CD4 T cell loss rates in human immunodeficiency virus-1 infection.

Additional progression markers for human immunodeficiency virus (HIV) infection are warranted. In this study we related antigen-specific responses in CD4(+) and CD8(+) T cells to CD38, reflecting chronic immune activation, and to CD4(+) T cell loss rates. Clones transiently expressing CD107a (CD8(+)) or CD154 (CD4(+)) in response to Gag, Env and Nef overlapping peptide pools were identified, along with their expression of the inhibitory programmed death-1 receptor (PD-1) in fresh peripheral blood mononuclear cells (PBMC) from 31 patients off antiretroviral treatment (ART). HIV-specific CD8(+) T cell responses dominated over CD4(+) T cell responses, and among CD8(+) responses, Gag and Nef responses were higher than Env-responses (P < 0.01). PD-1 on CD8(+) HIV-specific subsets was higher than CMV-specific CD8(+) cells (P < 0.01), whereas PD-1 on HIV-specific CD4(+) cells was similar to PD-1 on CMV-specific CD4(+) cells. Gag and Env CD8(+) responses correlated oppositely to the CD4 loss rate. Env/Gag CD8(+) response ratios, independently of PD-1 levels, correlated more strongly to CD4 change rates (r = -0.50 to -0.77, P < 0.01) than the total number of Gag-specific CD8(+) cells (r = 0.44-0.85, P < or = 0.02). The Env/Gag ratio performed better than CD38 and HIV-RNA in logistic regression analysis predicting CD4 change rate as a measure of progression. In conclusion, HIV-specific CD8(+)CD107a(+) Env/Gag response ratio was a stronger predictor for progression than CD38 and HIV-RNA. The Env/Gag ratio may reflect the balance between possibly beneficial (Gag) and detrimental (Env) CD8(+) T cell responses and should be explored further as a progression marker.

Human naturally occurring and adaptive regulatory T cells secrete high levels of leukaemia inhibitory factor upon activation.

Leukaemia inhibitory factor (LIF) is a member of the IL-6 cytokine family which signals through cognate receptors and activates target genes involved in survival, apoptosis, proliferation, differentiation and suppression of differentiation in different cell types. Binding of LIF to the LIFRalpha/gp130 receptor complex has been shown to activate the Janus kinase-signal transducer and activator of transcription 3 pathway. Here we show that activation of naturally occurring and adaptive regulatory T cells leads to increased LIF expression which is abrogated by cyclic adenosine monophosphate. Furthermore, the LIF receptors gp130 and LIFRalpha are upregulated on the surface of activated T cells and signal transducer and activator of transcription 3 phosphorylation is increased. Interestingly, LIF was not required for suppressive function but rather appeared to have a stimulatory effect on T cells that served to modulate and counteract immunosuppression by regulatory T cells.

Molecular architecture of signal complexes regulating immune cell function.

Signals transmitted via multichain immunoreceptors control the development, differentiation and activation of hematopoetic cells. The cytoplasmic parts of these receptors contain immunoreceptor tyrosine-based activation motifs (ITAMs) that upon phosphorylation by members of the Src tyrosine kinase family orchestrate a complex set of signaling events involving tyrosine phosphorylation, generation of second messengers like DAG, IP3 and Ca2+, activation of effector molecules like Ras and MAPKs and the translocation and activation of transcription factors like NFAT, API and NF-kB. Spatial and temporal organization of these signaling events is essential both to connect the receptors to downstream cascades as well as to control the functional outcome of the immune activation. Throughout this process control and fine-tuning of the different signals are necessary both for effective immune function and in order to avoid inappropriate or exaggerated immune activation and autoimmunity. This control includes modulating mechanisms that set the threshold for activation and reset the activation status after an immune response has been launched. One immunomodulating pathway is the cAMP-protein kinase A-Csk pathway scaffolded by a supramolecular complex residing in lipid rafts with the A kinase-anchoring protein (AKAP) ezrin, the Csk-binding protein PAG and a linker between the two, EBP50. Failure of correct scaffolding and loss of spatiotemporal control can potentially have severe consequences, leading to immune failure or autoimmunity. The clinical relevance of supramolecular complexes specifically organized by scaffolding proteins in regulating immune activity and the specter of genetic diseases linked to different signaling components suggest that protein-protein contact surfaces can be potential targets for drug intervention. It is also of interest to note that different pathogens have evolved strategies to specifically modulate signal integration, thereby rewiring the signal in a way beneficial for their survival. In addition to demonstrating the importance of different signal processes, these adaptations are elegant illustrations of the potential for drug targeting of protein assembly. This chapter reviews some of the important scaffolding events downstream of immunoreceptors with focus on signaling transduction through the T-cell receptor (TCR).

Spatiotemporal control of cAMP signalling processes by anchored signalling complexes.

Ligand-induced changes in cAMP concentration vary in duration, amplitude and extension into the cell. cAMP microdomains are shaped by adenylate cyclases that form cAMP as well as PDEs (phosphodiesterases) that degrade cAMP. Various extracellular signals converge on the cAMP/PKA (protein kinase A) pathway through ligand binding to GPCRs (G-protein-coupled receptors) and the cAMP/PKA pathway is therefore tightly regulated on several levels to maintain specificity in the multitude of signal inputs. AKAPs (A-kinase-anchoring proteins) target PKA to specific substrates and distinct subcellular compartments, providing spatial and temporal specificity for mediation of biological effects channelled through the cAMP/PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphoprotein phosphatases and cAMP-specific PDEs as well as components of other signalling pathways into multiprotein signalling complexes.

The molecular machinery for cAMP-dependent immunomodulation in T-cells.

cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I-Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR-CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP-PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, beta-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.

Compartmentalized cAMP signalling is important in the regulation of Ca(2+) cycling in the heart.

Co-ordinated myocyte handling of calcium is essential for efficient excitation-contraction coupling in the heart. The calcium cycling activity can be modulated by adrenergic stimulation and subsequent phosphorylation. Important functional consequences of phosphorylation include a greater influx of calcium through the voltage-dependent L-type Ca(2+) channel and a greater release of calcium from SR (sarcoplasmic reticulum) through the ryanodine R2 receptor. Furthermore, a more efficient reuptake through SERCA2 (sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase 2) is a result of phosphorylation of its regulatory protein phospholamban. Compartmentalized signalling is important in this signalling cascade, and A-kinase-anchoring proteins play a central role by providing a high level of specificity.

Expression of serotonin receptors 2B and 4 in human prostate cancer tissue and effects of their antagonists on prostate cancer cell lines.

OBJECTIVES: Overexpression of receptors to neuroendocrine (NE) cell products has been suggested to contribute to development of hormone-refractory prostate cancer (HRPC). In this study, we evaluated the expression of 5-HTR2B and 5-HTR4 in HRPC, and the effects of their antagonist on PC cell line growth. METHODS: Proteins and mRNA expression was determined by immunohistochemistry, western blot and RT-PCR. Growth inhibition of PC cell lines was determined in vitro using ELISA-BrdU proliferation assay and cell cycle was evaluated by flow cytometry. RESULTS: Immunostaining of 5-HTR2B was observed in low-grade and high-grade tumours, PIN and BPH cells, and in vascular endothelial cells, whereas 5-HTR4 was found predominantly in high-grade tumours. This result was confirmed by western blot analysis. At the mRNA level, 5-HTR4 mRNA was expressed in DU145 and LNCaP cells. Antagonists to both receptor subtypes inhibited proliferation of PC cells in a dose-dependent manner. CONCLUSIONS: The present result indicate that 5-HTRs are present at various tumour stages and that antagonists to these receptors can inhibit the proliferative activity of androgen-independent PC cell lines.

Identification, localization, and function in steroidogenesis of PAP7: a peripheral-type benzodiazepine receptor- and PKA (RIalpha)-associated protein.

Peptide hormones and cAMP acutely stimulate steroid biosynthesis by accelerating the transport of cholesterol into the mitochondria. The peripheral-type benzodiazepine receptor (PBR) has been shown to be an indispensable element of the cholesterol transport machinery. Using the yeast two-hybrid system and PBR as bait, we identified a protein that interacts with PBR, the PBR-associated protein PAP7. Using the regulatory subunit RIalpha of PKA as bait, we also isolated PAP7. Glutathione-S-transferase -PAP7 interacted with both the mitochondrial PBR and cytosolic PKA-RIalpha in MA-10 Leydig cells. PAP7 is a novel 52-kDa protein present in mouse, rat, and human tissues, and it has a major 3-kb mRNA transcript in all tissues examined. Immunohistochemical and in situ hybridization studies indicated that PAP7 is highly expressed in the gonads, adrenal, hippocampus, and distinct brain neuronal and glial populations. Overexpression of the full length PAP7 increased the hCG-induced steroid production. However, overexpression of a partial PAP7, which includes the PBR- and PKA-RIalpha-binding domains, inhibited the hormone-stimulated cholesterol transport and steroid synthesis. Treatment of MA-10 cells with oligonucleotides antisense to PAP7 also inhibited the hCG-stimulated steroid formation, suggesting that PAP7 is a functional element of the hormone-induced signal transduction cascade leading to steroidogenesis. PAP7 may function by targeting the PKA isoenzyme to organelles rich in PBR, i.e. mitochondria, where phosphorylation of specific protein substrates may induce the reorganization of PBR topography and function.

Cyclic AMP regulates expression of the RI alpha subunit of cAMP-dependent protein kinase through an alternatively spliced 5' UTR.

The present study examines novel mechanisms that regulate levels of the RI alpha subunit of cAMP-dependent protein kinase. We found that RI alpha protein is induced threefold by 8-(4-chlorophenyl)thio-cAMP in hormone responsive rat Sertoli cells, while total RI alpha mRNA is not correspondingly induced. Two RI alpha mRNA isoforms with different 5' untranslated sequences (RI alpha 1a and RI alpha 1b) are produced from the RI alpha gene in Sertoli cells. Deletion/mutation analysis of the cAMP-response-element-containing promoter upstream of the RI alpha exon 1b revealed that while mutation of the cAMP response element had no effects on cAMP-mediated induction, a 73-bp region of the RI alpha exon 1b itself conferred a fivefold to eightfold induction of reporter activity to homologous and heterologous promoters. The responsiveness of this region was dependent on a sense orientation downstream of the promoter start sites and had no effect on reporter mRNA, indicating that the cAMP-mediated induction occurs at the post-transcriptional level. Modeling of the RI alpha 1b 5' UTR secondary structure revealed a 5' CAP-proximal, strong stem-loop presenting an element similar to multiple start-site element downstream-1 (GCTCGG) in the loop region. RNA-EMSAs performed with the labeled RI alpha 1b 5' UTR showed stabilization of a protein/RNA complex in extracts from 8-(4-chlorophenyl)thio-cAMP stimulated Sertoli cells. This complex was abolished by mutation of the multiple start-site element downstream-1-like element. Our findings indicate that there is a cAMP-mediated induction of RI alpha expression at the post-transcriptional level, dependent on the 5' UTR of RI alpha 1b mRNA.

Regulation of anchoring of the RIIalpha regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIalpha: implications for chromosome dynamics at mitosis.

CDK1 phosphorylates the A-kinase regulatory subunit RIIalpha on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIalpha. Using an RIIalpha-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIalpha or an RIIalpha(T54E) mutant, we show that RIIalpha associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIalpha on T54. During interphase, both RIIalpha and RIIalpha(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIalpha, but not RIIalpha(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIalpha. Disrupting AKAP95-RIIalpha anchoring or depleting RIIalpha from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIalpha is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondensation in vitro, which can be rescued by addition of wild-type RIIalpha or an RIIalpha(T54D) mutant, but not RIIalpha(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIalpha constitutes a molecular switch controlling anchoring of RIIalpha to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.

CDK1-mediated phosphorylation of the RIIalpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIalpha from centrosomes at mitosis.

Protein kinase A regulatory subunit RIIalpha is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34(cdc2) kinase (CDK1) has been shown to phosphorylate RIIalpha on T54 and this has been proposed to alter the subcellular localization of RIIalpha. We have made stable transfectants from an RIIalpha-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIalpha (RIIalpha(T54E)). When expressed, RIIalpha detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIalpha(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIalpha is solubilized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N-terminal RII-binding domain of AKAP450, whereas small neutral residues do not change affinity detected by surface plasmon resonance. In addition, only RIIalpha(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIalpha(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIalpha transfectants. Our results suggest that T54 phosphorylation of RIIalpha by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.