Proteomic-based analysis of nuclear signaling: PLCbeta1 affects the expression of the splicing factor SRp20 in Friend erythroleukemia cells

An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).     

Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.

The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control.     

Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.

Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.     

Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase.

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.     

Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1)

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor beta(1) (TGFbeta(1)) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta did not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta did not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism of regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.     

Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase.

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.     

Integrin Signaling at the M/G1 Transition Induces Expression of Cyclin E

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21^cip1/Waf1 and p27^Kip1 is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.     

Human cyclin B3. mRNA expression during the cell cycle and identification of three novel nonclassical nuclear localization signals

Cyclins form complexes with cyclin-dependent kinases. By controlling activity of the enzymes, cyclins regulate progression through the cell cycle. A- and B-type cyclins were discovered due to their distinct appearance in S and G(2) phases and their rapid proteolytic destruction during mitosis. Transition from G(2) to mitosis is basically controlled by B-type cyclins. In mammals, two cyclin B proteins are well characterized, cyclin B1 and cyclin B2. Recently, a human cyclin B3 gene was described. In contrast to the expression pattern of other B-type cyclins, we find cyclin B3 mRNA expressed not only in S and G(2)/M cells but also in G(0) and G(1). Human cyclin B3 is expressed in different variants. We show that one isoform remains in the cytoplasm, whereas the other variant is translocated to the nucleus. Transport to the nucleus is dependent on three autonomous nonclassical nuclear localization signals that where previously not implicated in nuclear translocation. It had been shown that cyclin B3 coimmunoprecipitates with cdk2; but this complex does not exhibit any kinase activity. Furthermore, a degradation-resistant version of cyclin B3 can arrest cells in G(1) and G(2). Taken together with the finding that cyclin B3 mRNA is not only expressed in G(2)/M but is also detected in significant amounts in resting cells and in G(1) cells. This may suggest a dominant-negative function of human cyclin B3 in competition with activating cyclins in G(0) and the G(1) phase of the cell cycle.     

K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway

Phospholipase C β1 (PLCβ1) is known to play an important role in cell proliferation. Previous studies reported an involvement of PLCβ1 in G₀-G₁/S transition and G₂/M progression in Friend murine erythroleukemia cells (FELC). However, little has been found about its role in human models. Here, we used K562 cell line as human homologous of FELC in order to investigate the possible key regulatory role of PLCβ1 during cell proliferation of this human cell line. Our studies on the effects of the overexpression of both these isoforms showed a specific and positive connection between cyclin D3 and PLCβ1 in K562 cells, which led to a prolonged S phase of the cell cycle and a delay in cell proliferation. In order to shed light on this mechanism, we decided to study the possible involvement of protein kinases C (PKC), known to be direct targets of PLC signaling and important regulators of cell proliferation. Our data showed a peculiar decrease of PKCα levels in cells overexpressing PLCβ1. Moreover, when we silenced PKCα, by RNAi technique, in order to mimic the effects of PLCβ1, we caused the same upregulation of cyclin D3 levels and the same decrease of cell proliferation found in PLCβ1-overexpressing cells. The key features emerging from our studies in K562 cells is that PLCβ1 targets cyclin D3, likely through a PKCα-mediated-pathway, and that, as a downstream effect of its activity, K562 cells undergo an accumulation in the S phase of the cell cycle.     

Inhibition of Cdk1 by Alsterpaullone and Thioflavopiridol Correlates with Increased Transit Time from Mid G2 Through Prophase

Current models suggest that cyclin B1/cdk1 regulates the G2 to M transition and that its activity is maximal during the period from prophase to metaphase in mammalian cells. Although data are lacking, the idea that cyclin B1/cdk1 regulates the transit time from prophase to metaphase is reasonable. Development of small molecule inhibitors of cyclin dependent kinases (cdk’s) as cancer therapeutics presents an opportunity to evaluate the effects of inhibiting cdk’s in asynchronous cell populations. Analysis of cdk1 inhibitors is complicated by their ability to inhibit other cdk’s in vitro at higher concentrations. In this study we measured the effects of two cdk1 inhibitors on S, G2, and M transit for Hela cells and correlated these effects on cyclin B1/cdk1 and cyclin A/cdk2 activities. Dose responses demonstrate that low concentrations of both compounds inhibited the activity of cdk1 but not cdk2 in HeLa cells. The partial loss of cdk1 activity at low doses induced a prophase accumulation during a 3 h period and an increased transit time through mitosis. In addition, both inhibitors lengthened the G2 transit time with progressively greater effect on mid and late G2. High doses of both inhibitors increased the S phase time, which correlated with the inhibition of cdk2 activity. These results suggest that cdk1-cyclin activity is rate limiting for cell cycle progression during a period from mid G2 through prophase.     

Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate

Epidemiological, in vitro cell culture, and in vivo animal studies have shown that green tea or its constituent polyphenols, particularly its major polyphenol epigallocatechin-3-gallate (EGCG) may protect against many cancer types. In earlier studies, we showed that green tea polyphenol EGCG causes a G0/G1-phase cell cycle arrest and apoptosis of human epidermoid carcinoma (A431) cells. We also demonstrated that these effects of EGCG may be mediated through the inhibition of nuclear factor kappa B that has been associated with cell cycle regulation and cancer. In this study, employing A431 cells, we provide evidence for the involvement of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery during cell cycle deregulation by EGCG. As shown by immunoblot analysis, EGCG treatment of the cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, p16 and p18, (ii) downmodulation of the protein expression of cyclin D1, cdk4 and cdk6, but not of cyclin E and cdk2, (iii) inhibition of the kinase activities associated with cyclin E, cyclin D1, cdk2, cdk4 and cdk6. Taken together, our study suggests that EGCG causes an induction of G1-phase ckis, which inhibit the cyclin-cdk complexes operative in G0/G1 phase of the cell cycle thereby causing a G0/G1-phase arrest of the cell cycle, which is an irreversible process ultimately resulting in an apoptotic cell death. We suggest that the naturally occurring agents such as green tea polyphenols which may inhibit cell cycle progression could be developed as potent anticancer agents for the management of cancer.     

Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.     

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.     

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

Inhibition of cdk1 by alsterpaullone and thioflavopiridol correlates with increased transit time from mid G2 through prophase

Current models suggest that cyclin B1/cdk1 regulates the G2 to M transition and that its activity is maximal during the period from prophase to metaphase in mammalian cells. Although data are lacking, the idea that cyclin B1/cdk1 regulates the transit time from prophase to metaphase is reasonable. Development of small molecule inhibitors of cyclin dependent kinases (cdk's) as cancer therapeutics presents an opportunity to evaluate the effects of inhibiting cdk's in asynchronous cell populations. Analysis of cdk1 inhibitors is complicated by their ability to inhibit other cdk's in vitro at higher concentrations. In this study we measured the effects of two cdk1 inhibitors on S, G2, and M transit for Hela cells and correlated these effects on cyclin B1/cdk1 and cyclin A/cdk2 activities. Dose responses demonstrate that low concentrations of both compounds inhibited the activity of cdk1 but not cdk2 in HeLa cells. The partial loss of cdk1 activity at low doses induced a prophase accumulation during a 3 h period and an increased transit time through mitosis. In addition, both inhibitors lengthened the G2 transit time with progressively greater effect on mid and late G2. High doses of both inhibitors increased the S phase time, which correlated with the inhibition of cdk2 activity. These results suggest that cdk1-cyclin activity is rate limiting for cell cycle progression during a period from mid G2 through prophase.