Alberto M. Martelli

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)

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.

Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells

Nuclear lamins are the most abundant components of the nuclear lamina, a 10–50‐nm‐thick fibrous layer underlying the inner nuclear envelope membrane. Nevertheless, a number of recent investigations performed on epithelial and fibroblast cells have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nucleoskeleton. We have studied the subnuclear distribution of lamins A and B1 in human erythroleukemia cells by using immunoblotting analysis and immunofluorescent staining of fractionated nuclei. In intact cells and isolated nuclei, antibodies to lamins A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. However, when chromatin was removed by nuclease digestion and extraction with nonionic detergent or solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix or scaffold. Preferential localization of lamin A to the inner part of the nucleus was also demonstrated by the presence of the majority of lamin A in the solubilized inner nuclear network subfraction. In contrast, lamin B1 was mainly recovered in the fraction corresponding to the nuclear periphery. Double labeling experiments showed that lamin A, but not lamin B1, colocalized with coiled and GATA‐1 bodies. Thus, our results support the hypothesis that lamin A, but not lamin B1, may be a component of an internal nucleoskeleton in human erythroleukemia cells. J. Cell. Physiol. 178:284–295, 1999.

Rab3A and Rab3D Control the Total Granule Number and the Fraction of Granules Docked at the Plasma Membrane in PC12 Cells

Rab proteins are Ras‐like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno‐electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild‐type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.

Phosphatidylinositol 3-Kinase Translocates to the Nucleus of Osteoblast-Like MC3T3-E1 Cells in Response to Insulin-Like Growth Factor I and Platelet-Derived Growth Factor But Not to the Proapoptotic Cytokine Tumor Necrosis Factor α

Changes in the metabolism of nuclear inositides phosphorylated in the D3 position of the inositol ring, which may act as second messengers, mainly have been linked to cell differentiation. To clarify a possible role of this peculiar class of inositides also during cell proliferation and/or apoptosis, we have examined the issue of whether or not in the osteoblast‐like clonal cell line MC3T3‐E1 it may be observed an insulin‐like growth factor‐I (IGF‐I)‐ and platelet‐derived growth factor (PDGF)‐dependent nuclear translocation of an active phosphatidylinositol 3‐kinase (PI 3‐K). We found that both the growth factors increased rapidly and transiently both the amount and the activity of immunoprecipitable nuclear PI 3‐K. Intranuclear PI 3‐K exhibited a massive tyrosine phosphorylation on the p85 regulatory subunit. Moreover, by means of coimmunoprecipitation experiments, we showed the presence, in isolated nuclei, of the p110β catalytic subunit of PI 3‐K. Enzyme translocation was blocked by the specific PI 3‐K inhibitor LY294002. In contrast, intranuclear translocation of PI 3‐K did not occur in response to the proapoptotic cytokine tumor necrosis factor α (TNF‐α). IGF‐I was able to counteract the apoptotic stimulus of TNF‐α and this was accompanied by the intranuclear translocation of PI 3‐K. LY294002 inhibited both intranuclear translocation of PI 3‐K and the rescuing effect of IGF‐I. These findings strongly suggest that an important step in the signaling pathways that mediate both cell proliferation and survival is represented by the intranuclear translocation of PI 3‐K.

Insulin-like growth factor-I-dependent stimulation of nuclear phospholipase C-?1 activity in Swiss 3T3 cells requires an intact cytoskeleton and is paralleled by increased phosphorylation of the phospholipase

Swiss 3T3 mouse fibroblasts were exposed to 10 μM colchicine to disrupt microtubules, then stimulated with insulin‐like growth factor‐I. Immunoprecipitation experiments showed that insulin‐like growth factor‐I receptor and insulin receptor substrate‐1 were tyrosine phosphorylated to the same extent in both cells treated with colchicine and in those not exposed to the drug. Moreover, the activity of phosphatidylinositol 3‐kinase was not affected by incubation with colchicine. While in nuclei prepared from cells not exposed to colchicine it was possible to detect an insulin‐like growth factor‐I‐dependent increase in the mass of diacylglycerol, as well as stimulation of phospholipase C activity, no similar changes were observed in nuclei obtained from cells treated with colchicine. Activation of the nuclear phospholipase activity was paralleled by an increase of its phosphorylation. Immunofluorescent studies revealed that mitogen‐activated protein kinase did not translocate towards the nucleus when the cytoskeleton was depolymerized. These results show that in Swiss 3T3 cells some as yet unknown events necessary for the insulin‐like growth factor‐I‐dependent activation of nuclear polyphosphoinositide metabolism require the presence of an intact cytoskeleton and are situated down‐stream the activation of insulin receptor substrate‐1 and phosphatidylinositol 3‐kinase. Activation of nuclear phospholipase C‐β1 might be linked to its phosphorylation and translocation of mitogen‐activated protein kinase to the nucleus. J. Cell. Biochem. 72:339–348, 1999.

Behavior of nucleolar proteins during the course of apoptosis in camptothecin‐treated HL60 cells

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin‐treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti‐nucleolar antibodies seen in various pathological disorders. J. Cell. Biochem. 78:264–277, 2000.

Biochemical and morphological characterization of the nuclear matrix from apoptotic HL-60 cells.

We have characterized the nuclear matrix‐intermediate filament fraction from control and apoptotic HL‐60 cells. Apoptosis was induced by exposure to the topoisomerase I inhibitor, camptothecin. By means of two‐dimensional polyacrylamide gel electrophoresis, striking qualitative and quantitative differences were seen in the protein composition of the nuclear matrix–intermediate filament fraction obtained from apoptotic cells in comparison with controls. Western blotting analysis of apoptotic nuclear matrix proteins revealed degradation of some (topoisomerase IIα, SAF‐A) but not other (SATB1 and nucleolin) components. Moreover, immunofluorescent staining for typical matrix antigens (NuMA protein, lamin B, SC‐35) showed that in 35–40% of the structures prepared from apoptotic samples, marked changes in the subnuclear distribution of these proteins were present. Striking morphological differences between control and apoptotic samples were also detected at the ultrastructural level. These results demonstrate that both biochemical and morphological changes can be detected in the nuclear matrix prepared from apoptotic HL‐60 cells. J. Cell. Biochem. 72:35–46, 1999.

Subnuclear localization of S/MAR-binding proteins is differently affected by in vitro stabilization with heat or Cu2+

Abstract.The nuclear matrix, a proteinaceous entity thought to be a scaffolding structure that determines the higher order organization of eukaryotic chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions, but must rather be stabilized before the application of extracting agents such as high salt solutions or lithium diiodosalicylate. We have examined the effect of two widely used stabilization procedures on the localization of nuclear matrix proteins. Four individual polypeptides were studied, all of which are scaffold or matrix-associated region (S/MAR)-binding proteins: SATB1, SAF-A/hnRNP-U, NuMA , and topoisomerase II α. Nuclei were isolated from K562 human erythroleukemia cells in a buffer containing spermine, spermidine, KCl and EDTA, and the nuclear matrix or scaffold was obtained by extraction with lithium diiodosalicylate after stabilization by heat treatment (37° or 42°C) or incubation with Cu2+ ions. When the localization of individual proteins was determined by immunofluorescent staining and confocal scanning laser microscopy, markedly different consequences of the two stabilization strategies became evident, ranging from a total maintenance of the localization (NuMA and topoisomerase II α) to a marked redistribution (SATB1 and SAF-A/hnRNP-U). Our results seem to indicate that a reevaluation of stabilization protocols employed for the preparation of the nuclear matrix is desirable, especially by performing morphological controls.