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Biomedical subjects

C J Sherr

Publications and source records attributed to C J Sherr.

At least 73 records · Page 4Linked to original sources
PubMed1995

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.

Amino Acid Sequence↗
PubMed1995

Mammalian G1 cyclins and cell cycle progression.

Since their discovery in 1991, the D- and E-type cyclins, their associated catalytic subunits, and the molecules that regulate their kinase activity have been subjects of intense scrutiny by a rapidly expanding group of investigators. Part of the excitement stems from an emerging realization that these molecules define a final common pathway for growth factor-induced and antiproliferative signals during the cell cycle. As such, their study is helping to describe the biochemical pathways that determine the basis of late GI phase control, much as studies of growth factors, receptors, cytoplasmic signaling molecules, and responding transcription factors helped to define temporally earlier steps in the mitogenic process. In retrospect, it is neither surprising that there is a functional interplay between these cell cycle regulators and previously recognized tumor suppressor gene products (p53 and pRB) that are themselves engaged in GI phase checkpoint control, nor is it surprising that genetic perturbations affecting the functions of these genes contribute to cancer. It seems equally likely that further elucidation of the functions of these molecules will help to better explain such diverse processes as differentiation and apoptosis, which cooperatively depend on the cell cycle machinery for their proper execution.

Animals↗
PubMed1994

Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation.

Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme.

Animals↗
PubMed1994

The ins and outs of RB: coupling gene expression to the cell cycle clock.

Extracellular growth-stimulatory and -inhibitory signals govern the subunit assembly and activity of G1 cyclin-dependent kinases (cdks), which in turn can phosphorylate the retinoblastoma gene product, pRb, to cancel its growth-suppressive function. Hypophosphorylated forms of pRb, present only during the G1 phase, sequester target proteins including known transcription factors, but pRb phosphorylation late in G1 prevents these interactions and thus frees factors to alter the expression of genes required for entry into S phase. Although pRb can act as a regulator of the G1-S transition, its loss is tolerated by most cells, suggesting that its functions overlap with those of other regulators or are restricted to special circumstances under which cells exit the division cycle.

Journal Article↗
PubMed1994

Monoclonal antibodies to mammalian D-type G1 cyclins.

D-type cyclins are necessary and rate-limiting for G1 progression during the mammalian cell cycle. Cyclins D1, D2, and D3 are encoded by distinct genes and are expressed in proliferating cells in a lineage-specific manner. Monoclonal antibodies (mAbs) generated to bacterially produced recombinant D-type cyclins were able to react with the native proteins expressed in mammalian cells. One mouse and three rat mAbs immunoprecipitated cyclin D1 from mouse macrophages. Only rat mAbs reacted with human cyclin D1 and cross-reacted with cyclin D2 expressed in proliferating T lymphocytes and human tumor cell lines. A single rat mAb to cyclin D2 exhibited a pattern of reactivity reciprocal to that of rat mAbs to D1. Three rat mAbs reacted specifically with mouse or human cyclin D3, but did not cross-react with cyclins D1 or D2 from either species. Representative mAbs were useful for immunoblotting and detected D-type cyclins coprecipitating in complexes recovered with antiserum to cyclin-dependent kinase-4 (CDK4). Because these mAbs detect D-type cyclins in the nuclei of fixed permeabilized cells, they should prove useful in documenting cyclin overexpression in those human tumors in which the genes are amplified or are targets of specific chromosomal rearrangements.

Animals↗
PubMed1994

p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest.

Cell-cell contact and TGF-beta can arrest the cell cycle in G1. Mv1Lu mink epithelial cells arrested by either mechanism are incapable of assembling active complexes containing the G1 cyclin, cyclin E, and its catalytic subunit, Cdk2. These growth inhibitory signals block Cdk2 activation by raising the threshold level of cyclin E necessary to activate Cdk2. In arrested cells the threshold is set higher than physiological cyclin E levels and is determined by an inhibitor that binds to cyclin E-Cdk2 complexes. A 27-kD protein that binds to and prevents the activation of cyclin E-Cdk2 complexes can be purified from arrested cells but not from proliferating cells, using cyclin E-Cdk2 affinity chromatography. p27 is present in proliferating cells, but it is sequestered and unavailable to interact with cyclin E-Cdk2 complexes. Cyclin D2-Cdk4 complexes bind competitively to and down-regulate the activity of p27 and may thereby act in a pathway that reverses Cdk2 inhibition and enables G1 progression.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

D-type cyclin-dependent kinase activity in mammalian cells.

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.

3T3 Cells↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Growth factor-regulated G1 cyclins.

Mammalian D-type cyclins act as regulatory subunits of cyclin-dependent kinases (cdks) to form holoenzymes whose activities are both necessary and rate limiting for progression through the first gap phase (G1) of the cell cycle. D-type cyclins act as growth factor sensors to integrate receptor-mediated signals with the cell cycle machinery and to thereby enforce the decision of cells to enter their DNA synthetic (S) phase. Mitogenic and anti-proliferative stimuli affect the transcription of D-type cyclin genes and modulate both cyclin and cdk protein synthesis, their assembly into holoenzymes, post-translational modification, and serine/threonine kinase activity. In turn, the holoenzymes facilitate G1 exit by phosphorylating key cellular substrates, the retinoblastoma protein among them. Negative regulators of macrophage G1 progression, including cyclic AMP and the immunosuppressant rapamycin, extinguish the activity of G1 cyclin-dependent kinases by inducing novel polypeptide inhibitors that both inhibit the post-translational activation of cyclin-bound cdks and directly interfere with their catalytic functions. Our emerging understanding of G1 cyclins, their associated cdks, their proximal regulators, and their cdk substrates has now begun to provide a mechanistic understanding of how cells interpret mitogenic signals and make the decision to replicate their chromosomal DNA.

Animals↗
PubMed1993

Inhibition of granulocyte differentiation by G1 cyclins D2 and D3 but not D1.

Growth factor-induced signals govern the expression of three D-type cyclins, which, in turn, function as regulatory subunits of cyclin-dependent kinases (cdks) to control cell cycle transitions during the late G1 interval. 32D myeloid cells, which self-renew as uncommitted precursors in interleukin 3 (IL-3), express cyclins D2 and D3 (but not D1) in complexes with cdk4 and cdk2. When transferred to granulocyte colony-stimulating factor (G-CSF), 32D cells stop dividing and terminally differentiate to mature neutrophils. Cyclin D and cdk4 expression ceased as cells underwent growth arrest in G-CSF, but cdk2 levels were sustained. 32D cells engineered to ectopically express D-type cyclins exhibited contracted G1 intervals with a compensatory lengthening of S phase but remained IL-3 dependent for cell growth; those overexpressing cyclins D2 and D3 (but not D1) were unable to differentiate and died in G-CSF. Cyclin D2 mutants, which cannot efficiently bind to, or functionally interact with, the retinoblastoma protein (pRb) or its relatives (p107) did not block differentiation. Conversely, the introduction of a catalytically inactive cdk4 mutant into cells overexpressing cyclin D2 restored their G-CSF response. The persistence of cdk2 and its predilection to functionally interact with cyclins D2 and D3 rather than D1 might explain the specificity of the differentiation blockade.

Amino Acid Sequence↗
PubMed1993

Coamplification of the CDK4 gene with MDM2 and GLI in human sarcomas.

The 34-kilodalton cyclin-dependent kinase, p34cdk4, is a major catalytic subunit of mammalian D-type cyclins, which act during the G1 phase of the cell cycle to enforce the decision of cells to enter S phase. A murine complementary DNA clone was used to clone the cognate human CDK4 gene, which was localized to human chromosome 12, band q13, by fluorescence in situ hybridization. Because this chromosomal band contains the GLI and MDM2 genes, which are frequently amplified in human sarcomas, we analyzed CDK4 copy number and expression in a panel of sarcoma cell lines. An osteosarcoma cell line, OsACL, manifested a 25-fold increased copy number of CDK4, amplified concordantly with both GLI and MDM2, whereas a rhabdomyosarcoma cell line, SJRH30, was found to have an amplicon that included CDK4 and GLI but not MDM2. CDK4 mRNA and protein were overexpressed in both cell lines, and nucleotide sequencing analysis indicated that the gene had not sustained mutations. These observations provide the first evidence for amplification of a gene encoding a cell division cycle protein kinase, complement recent data indicating that genes encoding D-type cyclins are targets of chromosomal rearrangement and gene amplification in tumor cells, and suggest that CDK4 amplification might contribute to oncogenesis.

Base Sequence↗
PubMed1993

Functional interactions of the retinoblastoma protein with mammalian D-type cyclins.

The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity. By contrast, cyclins D2 and D3, but not D1, activated another such kinase, cdk2. Introduction of cyclin D2 and Rb into the Rb-deficient cell line SAOS-2 led to overt Rb hyperphosphorylation, whereas Rb, expressed alone or together with cyclin D1, remained unphosphorylated. Cyclin D2-dependent phosphorylation inhibited its binding to the transcription factor E2F and reversed the Rb G1 exit block in the cell cycle. Thus, all D-type cyclins do not function equivalently, and one of them plays a major role in reversing the cycle-blocking function of a known tumor suppressor.

Amino Acid Sequence↗
PubMed1993

Amplification of cyclin genes in colorectal carcinomas.

The genetic status of cyclin genes was examined in a panel of 47 colorectal carcinoma cell lines. Cyclin D2 was found to be amplified in one tumor and cyclin E in another. In each of the two cases, the amplified cyclin gene was overexpressed at the protein or mRNA level. Cyclin D1, previously shown to be amplified in breast and other tumors, was not amplified in these cancers. These data suggest that a variety of cyclin genes can play a role in human tumorigenesis and that cyclins D2 and E are particularly important in a subset of colorectal neoplasms.

Carcinoma↗
PubMed1993

Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.

The product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylated a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activated cdk4 enzyme activity, whereas cyclins A, B1, and E did not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bound pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induced pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yielded ternary complexes. The transcription factor E2F-1 also bound to pRb in insect cells, and coexpression of cyclin D-cdk4 complexes, but neither subunit alone, triggered pRb phosphorylation and prevented its interaction with E2F-1. The D-type cyclins may play dual roles as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates.

Adenosine Triphosphate↗