Luca M. Neri

The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system.

Lipid second messengers, particularly those derived from the polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in a plethora of cell functions. One of the best-characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt. Recent findings have implicated Akt in cancer progression because it stimulates cell proliferation and suppresses apoptosis. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid molecules are important components of signaling pathways operating within the nucleus. PI3Ks, their lipid products, and Akt have also been identified at the nuclear level. In this review, we shall summarize the most updated findings about these molecules in relationship with the nuclear compartment and provide an overview of the possible mechanisms by which they regulate important cell functions.

Translocation of Akt/PKB to the nucleus of osteoblast‐like MC3T3‐E1 cells exposed to proliferative growth factors

An active phosphatidylinositol 3‐kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P3 activated PKC, the ζ isozyme. We have examined the issue of whether or not in the osteoblast‐like clonal cell line MC3T3‐E1 there may be observed an insulin‐like growth factor‐I‐ (IGF‐I) and platelet‐derived growth factor‐ (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF‐I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S‐phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre‐treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation.

Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-ζ translocation to the nucleus of NGF-treated PC12 cells

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738–29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non‐canonical 3‐phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC‐ζ nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3‐kinase (PI 3‐K) activityand its product, phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5)P3]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3‐K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3‐K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P3. Maximal translocation of PKC‐ζ from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG‐sensitive, PKC‐α. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1‐O‐octadeyl‐2‐O‐methyl‐sn‐glycero‐3‐phosphocholine, was unable to abrogate nuclear translocation of the DAG‐insensitive PKC‐ζ. These data suggest that a nuclear increase in PI 3‐K activity and PtdIns(3,4,5)P3 production are necessary for the subsequent nuclear translocation of PKC‐ζ. Furthermore, they point to the likelihood that PKC‐ζ is a putative nuclear downstream target of PI 3‐K during NGF‐promoted neural differentiation.—Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3‐kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC‐ζ translocation to the nucleus of NGF‐treated PC12 cells. FASEB J. 13, 2299–2310 (1999)

Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-α

It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-α to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-α translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1to S-phase transition.

MULTIPLE BIOLOGICAL RESPONSES ACTIVATED BY NUCLEAR PROTEIN KINASE C

Protein kinase C is a family of serine‐threonine kinases that are physiologically activated by a number of lipid cofactors and are important transducers in many agonist‐induced signaling cascades. To date, 12 different isozymes of this kinase have been identified and are believed to play distinct regulatory roles. Protein kinase C was thought to reside in the cytosol in an inactive conformation and translocate to the plasma membrane upon cell activation by different stimuli. Nevertheless, a growing body of evidence has illustrated that this family of isozymes is capable of translocating to other cellular sites, including the nucleus. Moreover, it seems that some protein kinase C isoforms are resident within the nucleus. A wealth of data is being accumulated, demonstrating that nuclear protein kinase C isoforms are involved in the regulation of several critical biological functions such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we will discuss the most significant findings concerning nuclear protein kinase C which have been published during the past 5 years. J. Cell. Biochem. 74:499–521, 1999.

High-resolution Detection of Newly Synthesized DNA by Anti-Bromodeoxyuridine Antibodies Identifies Specific Chromatin Domains'

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.

Selective nuclear translocation of protein kinase C α in Swiss 3T3 cells treated with IGF-I, PDGF and EGF

To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent SvMs 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF‐I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan‐anti‐PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub‐cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the transiocation process, we have tested nine isozyme‐speeffic anti‐PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of α, βI, ε and ζ isoforms. In isolated nuclei from GF‐exposed cells only the α isozyme was detected: immunostaining was very intense after IGF‐I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC α and, because of the dramatic effect of IGF‐I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.

Temporal changes in intracellular distribution of protein kinase C in Swiss 3T3 cells during mitogenic stimulation with insulin-like growth factor I and bombesin: translocation to the nucleus follows rapid changes in nuclear polyphosphoinositides.

Using a polyclonal antibody raised against a synthetic peptide of the catalytic region of protein kinase C, we have carried out a combined immunocytochemical and immunochemical analysis to follow the subcellular localisation of this enzyme in response to mitogenic stimulation with insulin-like growth factor I and bombesin. These investigations show a time dependent translocation of protein kinase C from the cytoplasm to the nucleus since 5 min stimulation reaching a maximal effect after 45 min. These results show clearly that mitogen induced translocation of protein kinase C to the nucleus follows temporally the earlier changes in nuclear polyphosphoinositide metabolism previously demonstrated.

Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin.

We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3‐kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co‐immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G0/G1 phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34+/CD4−/CD7−), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.