The addressing fragment of mitogaligin: first insights into functional and structural properties.

Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31-53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.

Characterization of sinoatrial automaticity in Microcebus murinus to study the effect of aging on cardiac activity and the correlation with longevity.

Microcebus murinus, or gray mouse lemur (GML), is one of the smallest primates known, with a size in between mice and rats. The small size, genetic proximity to humans and prolonged senescence, make this lemur an emerging model for neurodegenerative diseases. For the same reasons, it could help understand how aging affects cardiac activity. Here, we provide the first characterization of sinoatrial (SAN) pacemaker activity and of the effect of aging on GML heart rate (HR). According to GML size, its heartbeat and intrinsic pacemaker frequencies lie in between those of mice and rats. To sustain this fast automaticity the GML SAN expresses funny and Ca2+ currents (If, ICa,L and ICa,T) at densities similar to that of small rodents. SAN automaticity was also responsive to ß-adrenergic and cholinergic pharmacological stimulation, showing a consequent shift in the localization of the origin of pacemaker activity. We found that aging causes decrease of basal HR and atrial remodeling in GML. We also estimated that, over 12 years of a lifetime, GML generates about 3 billion heartbeats, thus, as many as humans and three times more than rodents of equivalent size. In addition, we estimated that the high number of heartbeats per lifetime is a characteristic that distinguishes primates from rodents or other eutherian mammals, independently from body size. Thus, cardiac endurance could contribute to the exceptional longevity of GML and other primates, suggesting that GML's heart sustains a workload comparable to that of humans in a lifetime. In conclusion, despite the fast HR, GML replicates some of the cardiac deficiencies reported in old people, providing a suitable model to study heart rhythm impairment in aging. Moreover, we estimated that, along with humans and other primates, GML presents a remarkable cardiac longevity, enabling longer life span than other mammals of equivalent size.

L-Type Cav1.3 Calcium Channels Are Required for Beta-Adrenergic Triggered Automaticity in Dormant Mouse Sinoatrial Pacemaker Cells.

BACKGROUND: Sinoatrial node cells (SANC) automaticity is generated by functional association between the activity of plasmalemmal ion channels and local diastolic intracellular Ca2+ release (LCR) from ryanodine receptors. Strikingly, most isolated SANC exhibit a "dormant" state, whereas only a fraction shows regular firing as observed in intact SAN. Recent studies showed that ß-adrenergic stimulation can initiate spontaneous firing in dormant SANC, though this mechanism is not entirely understood. METHODS: To investigate the role of L-type Cav1.3 Ca2+ channels in the adrenergic regulation of automaticity in dormant SANC, we used a knock-in mouse strain in which the sensitivity of L-type Cav1.2 α1 subunits to dihydropyridines (DHPs) was inactivated (Cav1.2DHP-/-), enabling the selective pharmacological inhibition of Cav1.3 by DHPs. RESULTS: In dormant SANC, ß-adrenergic stimulation with isoproterenol (ISO) induced spontaneous action potentials (AP) and Ca2+ transients, which were completely arrested with concomitant perfusion of the DHP nifedipine. In spontaneously firing SANC at baseline, Cav1.3 inhibition completely reversed the effect of ß-adrenergic stimulation on AP and the frequency of Ca2+ transients. Confocal calcium imaging of SANC showed that the ß-adrenergic-induced synchronization of LCRs is regulated by the activity of Cav1.3 channels. CONCLUSIONS: Our study shows a novel role of Cav1.3 channels in initiating and maintaining automaticity in dormant SANC upon ß-adrenergic stimulation.

Regulation of the conserved 3'-5' exoribonuclease EXOSC10/Rrp6 during cell division, development and cancer.

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 processes and degrades RNA, regulates gene expression and participates in DNA double-strand break repair and control of telomere maintenance via degradation of the telomerase RNA component. EXOSC10/Rrp6 is part of the multimeric nuclear RNA exosome and interacts with numerous proteins. Previous clinical, genetic, biochemical and genomic studies revealed the protein's essential functions in cell division and differentiation, its RNA substrates and its relevance to autoimmune disorders and oncology. However, little is known about the regulatory mechanisms that control the transcription, translation and stability of EXOSC10/Rrp6 during cell growth, development and disease and how these mechanisms evolved from yeast to human. Herein, we provide an overview of the RNA- and protein expression profiles of EXOSC10/Rrp6 during cell division, development and nutritional stress, and we summarize interaction networks and post-translational modifications across species. Additionally, we discuss how known and predicted protein interactions and post-translational modifications influence the stability of EXOSC10/Rrp6. Finally, we explore the idea that different EXOSC10/Rrp6 alleles, which potentially alter cellular protein levels or affect protein function, might influence human development and disease progression. In this review we interpret information from the literature together with genomic data from knowledgebases to inspire future work on the regulation of this essential protein's stability in normal and malignant cells.

Impaired muscle regeneration and myoblast differentiation in mice with a muscle-specific KO of IGF-IR.

IGF-I and its receptor IGF-IR are seen as critical effectors of muscle hypertrophy, a notion recently questioned. Using MKR transgenic mice that express a dominant negative IGF-IR only in skeletal muscle, we have examined the role of the IGF-IR signaling in differentiation and repair of muscle fibers after damage-induced muscle regeneration. This process is impaired in MKR muscle, with incomplete regeneration, persistence of infiltrating cells and sustained expression of differentiation markers. Analysis of MKR and WT muscle-derived progenitor stem cells and myoblasts showed twice as many such cells in MKR muscle and an incomplete in vitro differentiation, that is, despite similar levels of myogenin expression, the level of fusion of MKR myoblasts was significantly reduced in comparison to WT myoblasts. These data show IGF-IR signaling is not only required at early hyperplasia stages of muscle differentiation, but also for late stages of myofiber maturation and hypertrophy.

ASAP is a novel substrate of the oncogenic mitotic kinase Aurora-A: phosphorylation on Ser625 is essential to spindle formation and mitosis.

Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to another and to prevent aneuploidy, which is mainly found in solid cancers. A correct mitotic spindle is necessary to accomplish such a process. Aurora kinases play critical roles in chromosome segregation and cell division; their deregulation impairs spindle assembly, checkpoint function and cell division causing chromosome mis-segregation. These kinases have been implicated in tumorigenesis. Aurora-A (AurA), in particular has been identified as a cancer-susceptibility gene, is overexpressed in a number of tumors and is required for G2/M transition and spindle assembly. ASAP is a novel spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that ASAP is a novel substrate of AurA kinase. We have identified serine 625 as the major phosphorylation site for AurA in vivo and localized the phosphorylated form of ASAP to centrosomes from late G2 to telophase, and around the midbody during cytokinesis. AurA depletion induces a proteasome-dependent degradation of ASAP. ASAP depletion induces spindle defects rescued by the expression of the phosphorylation-mimetic mutant ASAP-S625E and not by the non-phosphorylatable mutant ASAP-S625A. Microinjection of mono-specific S625 phospho-antibodies also impaired spindle formation and mitosis. These results strongly indicate that the phosphorylation of ASAP on S625 by AurA is required for bipolar spindle assembly and is essential for a correct mitotic progression. All together, these results suggest that we have identified a novel AurA substrate, pointing out ASAP as a new potential target for antitumoral drugs.

Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis.

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.

Metformin partially reverses the inhibitory effect of co-culture with ER-/PR-/HER2+ breast cancer cells on biomarkers of monocyte antitumor activity.

BACKGROUND: Immune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to MOs (monocytes) activity during their crosstalk with breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities, including cellular immunometabolism and protective redox signaling based-biomarkers, intracellular free calcium ions (ifCa2+), phagocytosis and co-operative cytokines (IFN-γ and IL-10) of autologous MOs before and during their interplay with primary ER-/PR-/HER2+ breast cancer cells. METHODS: Human primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET. RESULTS: MET downregulated breast cancer cell proliferation and phagocytosis, while having no significant effect on the ratio of phosphorylated Akt (p-Akt) to total Akt. Additionally, we observed that, in the absence of MET treatment, the levels of lactate dehydrogenase (LDH)-based cytotoxicity, catalase, ifCa2+, IL-10 and arginase activity were significantly reduced in co-cultures compared to levels in MOs cultured alone whereas levels of inducible nitric oxide synthase (iNOS) activity were significantly increased. In contrast, MET treatment reduced the effects measured in co-culture on the levels of LDH-based cytotoxicity, arginase activity, catalase, ifCa2+, and IFN-γ. MET also induced upregulation of both iNOS and arginase in MO cells, although the increase did not reach significant difference for iNOS activity. Moreover, MET induced a robust increase of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. Furthermore, MET markedly upregulated the levels of IFN-γ production and downregulated those of IL-10 in isolated MOs, while inducing a slight opposing up-regulation of IL-10 production in co-cultures. CONCLUSIONS: Our results show that the biomarkers of phenotypic functional activities of MOs are modified after co-culturing with primary human breast cancer cells. Treatment of co-cultures with MET resulted in increased release of antitumor cytokine IFN-γ and ifCa2+, and increased cell necrosis during breast cancer cells-MOs crosstalk.

Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding.

Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

Combination of metformin with sodium selenite induces a functional phenotypic switch of human GM-CSF monocyte-derived macrophages.

OBJECTIVES: We evaluated the effects of metformin (Met, 1,1­dimethylbiguanide hydrochloride) combined or not with sodium selenite (Ss, Na2SeO3) on the functional activities of LPS-activated GM-CSF monocyte-derived macrophages (GM-MDM). MATERIALS AND METHODS: Human GM-MDMs from three healthy donors were treated with Met or Ss alone, or with the combination of Met and Ss, and assayed for various biological activities and cytokines expression. RESULTS: Met alone and Ss alone had significantly different effects on phagocytosis and killing capacities and IL-ß production, but had similar effects on the downregulation of inducible nitric oxide synthase (iNOS) activity, relative nicotinamide adenine dinucleotide reduced (NADH) dehydrogenase (Complex I), intracellular free calcium ions (ifCa2+), and on the upregulation of arginase activity. Additionally, iNOS activity-to-arginase activity ratio was downregulated in Met or Ss treated-GM-MDMs, and, conversely, upregulated in GM-MDMs treated with Met + Ss in combination, indicating that arginase activity dominates that of iNOS when the two treatments are associated. Moreover, combination of Met with Ss significantly upregulated hydrogen peroxide (H2O2) production and phagocytic capacity, but significantly downregulated the production of IL-1ß, iNOS activity and killing capacity. On the contrary, we show that Met alone induced significant downregulation of phagocytic capacity and slight upregulation of killing capacity. Nevertheless, Ss seems to accentuate the effect of Met on the downregulation of NO production, as well as to reverse its effect on both phagocytic and killing capacities. On the other hand, all treatments induced a sharp decrease in relative levels of NADH dehydrogenase, and a marked decrease in the levels of ifCa2+. Finally, we found that GM-MDMs treated with Met or Ss, or Met combined with Ss exhibited different functional activation phenotypes, as indicated by the surface expression of co-stimulatory and cell activation and presentation molecules CD14, CD80, CD86 and HLA-DR. CONCLUSIONS: Our results demonstrated that Met/Ss combination can play an important role in the modulation of functional activities of human LPS-activated GM-MDMs. Additionally, the overall effects of Met and the induction of "M2" GM-MDMs-associated arginase could be influenced by its combination with Ss.

Akt2 is implicated in skeletal muscle differentiation and specifically binds Prohibitin2/REA.

Akt1 and Akt2 are the major isoforms of Akt expressed in muscle cells and muscle tissue. We have performed siRNA silencing of Akt1 and Akt2 in C2 myoblasts to characterize their specific implication in muscle differentiation. Whereas silencing Akt2, and not Akt1, inhibited cell cycle exit and myoblast differentiation, Akt2 overexpression led to an increased proportion of differentiated myoblasts. In addition, we demonstrate that Akt2 is required for myogenic conversion induced by MyoD overexpression in fibroblasts. We show Akt2, but not Akt1, binds Prohibitin2/Repressor of Estrogen Activator, PHB2/REA, a protein recently implicated in transcriptionnal repression of myogenesis. Co-immunoprecipitation experiments on endogenous proteins showed the Akt2-REA complex does not contain Prohibitin1. We have analyzed expression and localization of PHB2/REA during proliferation and differentiation of mouse and human myoblasts. PHB2/REA shows punctated nuclear staining which partially co-localizes with Akt2 in differentiated myotubes and PHB2 levels decrease at the onset of myogenic differentiation concomitant with an increase in Akt2. There appears to be an inverse correlation between Akt2 and PHB2 protein levels where cells silenced for Akt2 expression show increased level of PHB2/REA and overexpression of Akt2 resulted in decreased Prohibitin2/REA. Taken together, these results, along with our previous observations, clearly show that Akt2 and not Akt1 plays a major and early role in cell cycle exit and myogenic differentiation and this function involves its specific interaction with PHB2/REA.

Macromolecular uptake is a spontaneous event during mitosis in cultured fibroblasts: implications for vector-dependent plasmid transfection.

The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be "vectorized" rather than acting as true vectors.

Functional cdc25C dual-specificity phosphatase is required for S-phase entry in human cells.

In view of the common regulatory mechanism that induces transcription of the mitotic phosphatase cdc25C and cyclin A at the beginning of S-phase, we investigated whether cdc25C was required for S-phase transit. Here, we show that in both nontransformed human fibroblasts and HeLa cells, cdc25C protein levels significantly increased concomitant with S-phase onset and cyclin A synthesis. Activity measurements on immunoprecipitates from synchronized HeLa cells revealed a sharp rise in cdc25C-associated phosphatase activity that coincided with S-phase. Microinjection of various antisense-cdc25C molecules led to inhibition of DNA synthesis in both HeLa cells and human fibroblasts. Furthermore, transfection of small interfering RNA directed against cdc25C specifically depleted cdc25C in HeLa cells without affecting cdc25A or cdc25B levels. Cdc25C RNA interference was also accompanied by S-phase inhibition. In cells depleted of cdc25C by antisense or siRNA, normal cell cycle progression could be re-established through microinjection of wild-type cdc25C protein but not inactive C377S mutant protein. Taken together, these results show that cdc25C not only plays a role at the G2/M transition but also in the modulation of DNA replication where its function is distinct from that of cdc25A.

MyoD distal regulatory region contains an SRF binding CArG element required for MyoD expression in skeletal myoblasts and during muscle regeneration.

We show here that the distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, bound two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppressed binding of both SRF and YY1 complexes and abolished DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppressed MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and beta-galactosidase was induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter was no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration.

Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy.

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.

Adult muscle-derived stem cells engraft and differentiate into insulin-expressing cells in pancreatic islets of diabetic mice.

BACKGROUND: Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of nonadherent muscle-derived stem cells (MDSC) from adult mouse muscle to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate for beta-cell deficiency. RESULTS: In vitro, cultured MDSC spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter. Differentiated clusters of beta-like cells co-expressed insulin with the transcription factors Pdx1, Nkx2.2, Nkx6.1, and MafA, and secreted significant levels of insulin in response to glucose challenges. In vivo, undifferentiated MDSC injected into streptozotocin (STZ)-treated mice engrafted within 48 h specifically to damaged pancreatic islets and were shown to differentiate and express insulin 10-12 days after injection. In addition, injection of MDSC into hyperglycemic diabetic mice reduced their blood glucose levels for 2-4 weeks. CONCLUSION: These data show that MDSC are capable of differentiating into mature pancreatic beta islet-like cells, not only upon culture in vitro, but also in vivo after systemic injection in STZ-induced diabetic mouse models. Being nonteratogenic, MDSC can be used directly by systemic injection, and this potential reveals a promising alternative avenue in stem cell-based treatment of beta-cell deficiencies.

Type II PKAs are anchored to mature insulin secretory granules in INS-1 ß-cells and required for cAMP-dependent potentiation of exocytosis.

Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIß PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting ß-cells. Ultrastructural analysis of native rodent ß-cells confirmed in vivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIß PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the ß-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis.

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase.

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.

Characterization of the Akt2 domain essential for binding nuclear p21cip1 to promote cell cycle arrest during myogenic differentiation.

The binding of the cdk inhibitor p21cip1 to Akt2 in the nucleus is an essential component in determining the specific role of Akt2 in the cell cycle arrest that precedes myogenic differentiation. Here, through a combination of biochemical and cell biology approaches, we have addressed the molecular basis of this binding. Using amino-terminal truncation of Akt2, we show that p21cip1 binds at the carboxy terminal of Akt2 since deletion of the first 400 amino acids did not affect the interaction between Akt2 and p21cip1. Pull down using carboxy terminal-truncated Akt2 protein revealed the importance of the region between amino acids 400 and 445 for the binding to p21cip1. Since Akt2_400-445 and Akt2_420-445 peptides could both bind p21cip1, this refines the binding domain on Akt2 between amino acids 420 and 445. In order to confirm these data in living cells, we developed a protocol to synchronize myoblasts at the cell cycle exit point when p21cip1 expression is induced by MyoD before myogenic differentiation. When a synthetic Akt2 peptide spanning the region (410-437) was microinjected in p21-expressing myoblasts, p21cip1 no longer localized exclusively in the nucleus, instead being redistributed throughout the cell, thus showing that injected peptide 410-437 acts to compete with the binding of endogenous Akt2 to p21cip1. Taken together, our data suggest that this 27 amino acid sequence on Akt2 is necessary and sufficient to bind p21cip1 both in vitro and in living cells.