Swimming against the tide: progress and challenges in our understanding of colicin translocation

Colicins are folded protein toxins that face the formidable task of translocating across one or both of the Escherichia coli cell membranes in order to induce cell death. This translocation is achieved by parasitizing host proteins. There has been much recent progress in our understanding of the early stages of colicin entry, including the binding of outer-membrane nutrient transporters and porins and the subsequent recruitment of periplasmic and inner-membrane proteins that, together, trigger translocation. As well as providing insights into how these toxins enter cells, these studies have highlighted some surprising similarities in the modes of action of the systems that colicins subvert.     

Current understanding of glucose transporter 4 expression and functional mechanisms

Glucose is used aerobically and anaerobically to generate energy for cells. Glucose transporters (GLUTs) are transmembrane proteins that transport glucose across the cell membrane. Insulin promotes glucose utilization in part through promoting glucose entry into the skeletal and adipose tissues. This has been thought to be achieved through insulin-induced GLUT4 translocation from intracellular compartments to the cell membrane, which increases the overall rate of glucose flux into a cell. The insulin-induced GLUT4 translocation has been investigated extensively. Recently, significant progress has been made in our understanding of GLUT4 expression and translocation. Here, we summarized the methods and reagents used to determine the expression levels of Slc2a4 mRNA and GLUT4 protein, and GLUT4 translocation in the skeletal muscle, adipose tissues, heart and brain. Overall, a variety of methods such real-time polymerase chain reaction, immunohistochemistry, fluorescence microscopy, fusion proteins, stable cell line and transgenic animals have been used to answer particular questions related to GLUT4 system and insulin action. It seems that insulin-induced GLUT4 translocation can be observed in the heart and brain in addition to the skeletal muscle and adipocytes. Hormones other than insulin can induce GLUT4 translocation. Clearly, more studies of GLUT4 are warranted in the future to advance of our understanding of glucose homeostasis.     

Gene amplification in a human osteosarcoma cell line results in the persistence of the original chromosome and the formation of translocation chromosomes.

Although gene amplification, a process that is markedly enhanced in tumor cells, has been studied in many different cell systems, there is still controversy about the mechanism(s) involved in this process. It is still unclear what happens to the DNA sequences that become amplified, whether they remain present at their original location (conservative gene amplification) or whether gene amplification necessarily results in a deletion at the original location (non-conservative gene amplification). We have studied gene amplification in a human osteosarcoma cell line, starting from a cell clone which contains only one copy of a plasmid integrate. Independent amplificants, originating from this clone and containing elevated plasmid copy numbers, were isolated and analyzed. Based on previous observations, encompassing the persistence of single-copy DNA sequences besides amplified DNA sequences clustered at a different location in the independent amplificants, we proposed an amplification pathway including a local duplication step and transposition of the duplicated DNA to other chromosomal positions. Now we have extended our study to more independent amplificants. We prove that the single-copy plasmid-containing chromosomes in the different amplificants and the single-copy plasmid-containing chromosome in the original parental cell clone are indeed identical, namely a translocation chromosome composed of at least three parts of which two originate from chromosomes 14 and 17. We show that the unit of amplification and the unit of the proposed transposition event are at least 1.5 Mb. We also demonstrate that the amplified DNA sequences, present at genomic locations other than the original single-copy DNA sequences, are preferentially associated with chromosome 16. We find that the amplified DNA sequences are often located at or near a site of chromosome translocation involving chromosome 16. In one cell clone we detect the amplified DNA sequences in most of the cells to be located within a complete chromosome 16 while in a minority of cells the amplified sequences are located at or near a breakpoint on a translocation chromosome 16. This indicates that this amplification region is highly unstable and frequently gives rise to translocation events.     

EspA filament-mediated protein translocation into red blood cells

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria–RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria–RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.     

A point mutation of proline 308 in diphtheria toxin B chain inhibits membrane translocation of toxin conjugates

Diphtheria toxin (DT) is a soluble protein that translocates across hydrophobic lipid bilayers in response to low pH. The translocation activity of DT has been localized to the 40-kDa toxin B chain and can be expressed independently of the C-terminal receptor binding site. Buried hydrophobic domains in DT are thought to participate in the membrane translocation process. We have identified a mutant form of DT, CRM 102, that has a point mutation at position 308 (Pro----Ser) within one of these hydrophobic domains. CRM 102 conjugated to a monoclonal antibody against the T cell receptor, the transferrin receptor, or transferrin itself is approximately 10-fold less toxic than native DT or a control DT mutant, CRM 103, linked to the same binding moieties. Direct measurement of membrane translocation activity by exposure of cells to low extracellular pH demonstrates that CRM 102 conjugates express only 10% of the translocation activity of the control toxin conjugates. However, when CRM 102 or 102 conjugates bind and kill cells via the DT receptor, no reduction in membrane translocation activity is observed. The defect in CRM 102 is not evident in the presence of 20 mM NH4Cl. The defect in translocation also has no effect on the ratio of the lag time before protein synthesis inhibition begins to the rate of protein synthesis inhibition. Thus, the proline-serine substitution at position 308 disrupts the membrane translocation process and distinguishes between two routes of DT entry: DT receptor-mediated entry and entry mediated by alternate receptors.     

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.     

Chromosomes and carcinogenesis. Study on the evolution of an epithelial cell line of porcine origin

The PFT cell line was established in 1969 from diploid cells of the inner lining of a uterine tube of a 2 year-old sow and has been continuously subcultured more than 500 times over a decade. Three chromosomal rearrangements have occurred during this time. The first translocation was shown at the 100th passage with the concomitant and spontaneous release of an endogeneous type C virus. The second translocation was observed at the 290th passage along with the appearance of gap junctions and the induction of malignant tumors in athymic nude mice following the inoculation of PFT cells. The third translocation was found towards the 470th passage with the simultaneous appearance of annulate lamellae. Since the translocations were accompanied by the spontaneous release of a retrovirus and then by malignancy of PFT cells when inoculated in athymic nude mice, it is likely that the chromosomal abnormalities are associated with the viral carcinogenesis of the cell line. The third translocation appears to confirm the perenniality of the multistep evolution hypothesis of malignancy.     

Natural phosphatidylcholine is actively translocated across the plasma membrane to the surface of mammalian cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.     

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.     

Cisplatin resistance is associated with deregulation in protein kinase C-delta

Proteolytic activation of protein kinase C (PKC)-delta has been associated with cell death induced by the DNA damaging agent cisplatin. In the present study, we have examined if PKCdelta is affected when cells acquire resistance to cisplatin. The level of PKCdelta was elevated in cisplatin-resistant HeLa (HeLa/CP) cells compared to parental HeLa cells. Prolonged cellular exposure to the PKC activator phorbol-12,13-dibutyrate (PDBu), caused downregulation of PKCdelta in HeLa cells but not in HeLa/CP cells. Treatment of HeLa cells with PDBu resulted in the translocation of PKCdelta from the cytosol to the membrane but it failed to induce PKCdelta translocation in HeLa/CP cells. PDBu, however, induced translocation and downregulation of PKCalpha in both HeLa and HeLa/CP cells. The ability of PDBu to enhance cisplatin-induced cell death was attenuated in cisplatin-resistant HeLa cells. Thus, a deregulation in PKCdelta was associated with reduced cellular sensitivity to cisplatin.     

Breaching biological barriers: protein translocation domains as tools for molecular imaging and therapy

The lipid bilayer of a cell presents a significant barrier for the delivery of many molecular imaging reagents into cells at target sites in the body. Protein translocation domains (PTDs) are peptides that breach this barrier. Conjugation of PTDs to imaging agents can be utilized to facilitate the delivery of these agents through the cell wall, and in some cases, into the cell nucleus, and have potential for in vitro and in vivo applications. PTD imaging conjugates have included small molecules, peptides, proteins, DNA, metal chelates, and magnetic nanoparticles. The full potential of the use of PTDs in novel in vivo molecular probes is currently under investigation. Cells have been labeled in culture using magnetic nanoparticles derivatized with a PTD and monitored in vivo to assess trafficking patterns relative to cells expressing a target antigen. In vivo imaging of PTD-mediated gene transfer to cells of the skin has been demonstrated in living animals. Here we review several natural and synthetic PTDs that have evolved in the quest for easier translocation across biological barriers and the application of these peptide domains to in vivo delivery of imaging agents.     

A t(4;22) in a meningioma points to the localization of a putative tumor-suppressor gene.

Cytogenetic analysis of meningioma cells from one particular patient (MN32) displayed the stem-line karyo-type 45, XY, -1, 4p+, 22q-, 22q+, which thus had rearrangements of both chromosomes 22. The 22q+ marker appeared as a dicentric: 22 pter----q11::1p11----qter. The reciprocal product of this translocation has presumably been lost because it lacked a centromere. The 22q- chromosome also appeared to have lost sequences distal to band q11. We assumed that this marker could have been the result of a reciprocal translocation between chromosomes 4 and 22. To investigate the 4p+ and 22q- chromosomes in more detail, human-hamster somatic cell hybrids were constructed that segregated the 22q- and 4p+ chromosomes. Southern blot analysis with DNA from these hybrids showed that sequences from 22q were indeed translocated to 4p+ and that reciprocally sequences from 4p were translocated to 22q-, demonstrating a balanced t(4;22)(p16;q11). On the basis of these results we presume that in this tumor a tumor-suppressor gene is deleted in the case of the 22q+ marker and that the t(4;22) disrupts the second allele of this gene. The latter translocation was mapped between D22S1 and D22S15, a distance of 1 cM on the linkage map of this chromosome. The area in which we have located the translocation is within the region where the gene predisposing to neurofibromatosis 2 has been mapped.     

Examining the mechanism of Erk nuclear translocation using green fluorescent protein

In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2.     

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides.Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane.In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.     

Effects of interferon-beta on the translocation rate and stationary time in human fibroblasts in culture

The rate of translocation and the percent of the time that cells are stationary have been measured by computer-assisted time-lapse cinemicrography in over 1,000 freshly planted human foreskin fibroblasts (FS-4 cell strain) for periods of up to a week and the effects of interferon-beta (IFN-beta) on these parameters have been determined. Cells were planted at 2.5 X 10(3) cells/cm2 in Eagle's minimal essential medium (MEM) with 10% fetal bovine serum (FBS). Frames were taken every 2 or 4 minutes and data were collected on both cell location and cell division as a function of time. After planting FS-4 cells require approximately 48 hr to reach maximum motility both with respect to the translocation rate when moving and percent time cells are moving. Recombinant human INF-beta (800 mu/ml) caused a marked increase in the fraction of time cells were stationary and a decrease of lesser magnitude in the translocation rate, as quantitated during the period during which the stationary fraction for control cells was at a minimum. IFN-beta also decreased the rate of cell proliferation, without any evidence of degeneration or death of cells. Our results contribute new evidence that the fraction of time cells spend moving directionally is an important determinant of their locomotory behavior and that this determinant is responsive to modulation by cytokines.     

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.     

Molecular cloning and characterization of grancalcin, a novel EF-hand calcium-binding protein abundant in neutrophils and monocytes.

A novel EF-hand Ca(2+)-binding protein we have called grancalcin has been identified and characterized. This protein is particularly abundant in neutrophils and monocytes, with relatively small amounts in lymphocytes. The cDNA for this protein has been cloned and sequenced. The sequence predicts that the protein is composed of 217 amino acids, with a molecular mass of 24,010 daltons. It contains four EF-hand calcium-binding motifs and exhibits strong homology to sorcin, one of two proteins overexpressed in multidrug-resistant cells whose function is unknown. There are potentially one phosphorylation and two glycosylation sites. The 1.65-kilobase mRNA is detected in bone marrow and is present in neutrophils, monocytes, macrophages, B and T lymphocytes, and the promyelocytic cell line HL60s. The protein displays a Ca(2+)-dependent translocation to the granules and plasma membrane of neutrophils, suggesting that it might play an effector role in the specialized functions of these cells.     

CagI is an essential component of the Helicobacter pylori Cag type IV secretion system and forms a complex with CagL

Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly.     

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)P(3) activated PKC, the zeta 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.