FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic fflament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleotides. A lethal ftsZ mutation, ftsZ3 (Rsa), that leads to an amino acid alteration in this homologous segment decreased GTP binding and hydrolysis, suggesting that interaction with GTP is essential for ftsZ function.FtsZ is an essential cell division protein in Escherichia coli (1-3). An increase in FtsZ stimulates cell division in the form of a minicell phenotype (4), whereas a small decrease in FtsZ leads to a decrease in division activity (1, 5). In addition, FtsZ acts earliest in the division pathway among known division proteins (6, 7) and is the apparent target of two division inhibitors: SulA, a component ofthe SOS response (8,9), and MinCD, a component of the min system (10, 11). By using immunoelectron microscopy, Bi and Lutkenhaus (12) demonstrated that the FtsZ protein in E. coli is localized at the division site in a ring structure designated the FtsZ ring. This localization of FtsZ into a ring that is near the cytoplasmic membrane at midcell is under cell cycle control. The FtsZ ring appears at midcell before invagination is visible, indicating that it is an early step in division. As division proceeds, the FtsZ ring decreases in diameter at the leading edge of the invagination. After completion of division, the FtsZ disassembles and is not associated with the new cell pole.The discovery of the FtsZ ring allows questions about cell division to be rephrased and more specifically stated. These questions include: (i) what is the cell cycle signal for formation of the FtsZ ring, (ii) how is the ring localized, and (iii) what is its function? In addition, questions can be asked about the structure of the ring and how the ring is assembled and disassembled. In their model for the functional role of FtsZ in cell division, Bi and Lutkenhaus (12) proposed that FtsZ is a cytoskeletal element that self-assembles into the ring structure to initiate division and then disassembles upon completion ofdivision. The observed dynamic localization of FtsZ is reminiscent of tubulin and actin in eukaryotic cells. Both of these eukaryotic proteins use a nucleoside triphosphate as a cofactor in the assembly process; actin uses ATP (13) and tubulin uses GTP (14). Interestingly, FtsZ contains a 7-amino acid segment (GGGTGTG) that is homologous to a segment found in tubulin (GGGTGSG) that is believed to be involved in the interaction of tubulin with guanine nucleotides (15, 16). As a first step in determining whether FtsZ might fall into this category of filament-forming proteins, we purified FtsZ and have examined its interaction...

TheftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies offtsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by X16-2, even though this bacteriophage can complement the thermosensitiveftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.

Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of theftsZ,fts4, orftsQ genes. We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ. Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA. Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ. In addition, although wild-type strains were relatively insensitive to overexpression ofJftQ, we observed that cell division was sensitized toftsQ overexpression in ftsl,ftL4, andftsZ mutants. Among these, theftsI mutant was the most sensitive. These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.

Transforming growth factor–β (TGF-β) inhibits cell proliferation, and acquisition of TGF-β resistance has been linked to tumorigenesis. A genetic screen was performed to identify complementary DNAs that abrogated TGF-β sensitivity in mink lung epithelial cells. Ectopic expression of murine double minute 2 rescued TGF-β–induced growth arrest in a p53-independent manner by interference with retinoblastoma susceptibility gene product (Rb)/E2F function. In human breast tumor cells, increased MDM2 expression levels correlated with TGF-β resistance. Thus, MDM2 may confer TGF-β resistance in a subset of tumors and may promote tumorigenesis by interference with two independent tumor suppressors, p53 and Rb.

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A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.

CDC37 was originally identified as a Start gene in budding yeast and has been shown to be required for association of CDC28 with cyclins. The exact functional mechanism by which CDC37 promotes this association, however, remains unknown. CDK4 is a cyclin D-dependent kinase that controls progression through G 1 of the mammalian cell cycle. We have detected a specific association of CDK4 with the molecular chaperon HSP90 and a 44-kDa protein that we identify as mammalian CDC37. A physical interaction between CDC37 and CDK4 suggests that CDC37 may regulate the mammalian cell cycle through a direct effect on CDK4. Association of CDK4 with both CDC37 and HSP90 may also imply a mechanistic link between the functions of CDC37 and HSP90.In mammalian cells, ordered activation of cyclin-dependent kinases (CDKs) 1 governs the progression through different phases of the cell cycle (1-4). CDK4 is one of the CDKs that control the G 1 -to-S phase transition. Activation of CDK4 requires binding to one of the D-type cyclins (D1, D2, or D3) (5) and phosphorylation by the CDK-activating kinase (CAK) (6). As cells enter the cycle from quiescence (G 0 ) in response to growth signals, CDK4 and cyclin D are synthesized and assemble into CDK4-cyclin D complexes. The assembly of CDK4-cyclin D complexes may also require an as yet unidentified upstream regulatory factor(s), since complex assembly in cells with ectopically expressed CDK4 and cyclin D is still dependent on mitogenic signals (7). Although required for the activation of CDK4, phosphorylation by CAK appears not needed for the assembly of CDK4-cyclin D complexes (6). Therefore, although much has been known about the functional mechanism and regulation of CDKs and their cyclin partners, new regulatory proteins and regulation pathways remain to be uncovered.In order to identify other proteins involved in CDK4 regulation, we analyzed CDK4 complexes in various mammalian cells by an approach employed in our previous studies (8 -11). We report the discovery of a direct association of CDK4 with mammalian CDC37, a protein potentially essential for activation of CDK4. EXPERIMENTAL PROCEDURESCell Lines and Antibodies-S6, WMN, and ML-1 cells were grown in RPMI 1640 containing 10% fetal bovine serum. NIH3T3 (ATCC CRL 1658) cells were grown in DMEM supplemented with 10% calf serum. The S6 is a subclone of mouse myeloid cell line M1 (13). WMN, a human Burkitt's lymphoma cell line, was kindly provided by Dr. Patrick M. O'Connor (National Cancer Institute, Bethesda, MD). Ml-1, a human myeloid cell line, was obtained from cell culture facilities at Cold Spring Harbor Laboratory (Cold Spring Harbor, NY). The antibody used to immunoprecipitate CDK4 from mouse cells was purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA: catalog number sc-260). It was raised against a peptide at the carboxyl terminus of CDK4 of mouse origin. The antibody used to immunoprecipitate CDK4 from human cells has been described (8). The anti-HSP90 monoclonal antibody was purchased from StressGen Biotechnologie...

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.