Biomedical subjects

J A Diehl

Publications and source records attributed to J A Diehl.

At least 19 recordsLinked to original sources

PubMed2006

AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Cell Differentiation↗

PubMed2006

Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1.

Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.

Alleles↗

PubMed2006

Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma.

Mantle cell lymphoma (MCL) is a B-cell lymphoma characterized by overexpression of cyclin D1 due to the t(11;14) chromosomal translocation. While expression of cyclin D1 correlates with MCL development, expression of wild-type (WT) cyclin D1 transgene in murine lymphocytes is unable to drive B-cell lymphoma. As cyclin D1 mutants that are refractory to nuclear export display heighten oncogenicity in vitro compared with WT D1, we generated mice expressing FLAG-D1/T286A, a constitutively nuclear mutant, under the control of the immunoglobulin enhancer, Emu. D1/T286A transgenic mice universally develop a mature B-cell lymphoma. Expression of D1/T286A in B lymphocytes results in S phase entry in resting lymphocytes and increased apoptosis in spleens of young premalignant mice. Lymphoma onset correlates with perturbations in p53/MDM2/p19Arf expression and with BcL-2 overexpression suggesting that alterations in one or both of these pathways may contribute to lymphoma development. Our results describe a cyclin D1-driven model of B-cell lymphomagenesis and provide evidence that nuclear-retention of cyclin D1 is oncogenic in vivo.

Animals↗

PubMed2006

Cyclin D1 and p21 is elevated in the giant cells of giant cell tumors.

Alterations of cell cycle regulatory proteins, especially those that regulate G1 to S transition, have been implicated in the pathogenesis of a wide variety of human tumors. In previous studies we showed that that there is overexpression of cyclin D1 protein predominately in the giant cell component of giant cell tumors of bone. The purpose of this study was to investigate the mechanisms that may be responsible for cyclin D1 accumulation in giant cell tumors. Giant cell tumors have high levels of cyclin D1 mRNA and the giant cell-enriched population of these tumors have significantly more mRNA and protein expression of cyclin D1 than the mononuclear cell population. The giant cells also expressed higher levels of p21 protein and more p21 bound to cyclin D1 than the mononuclear cells. It is possible that p21 may be contributing to the cyclin D1 accumulation that occurs in the giant cells and perhaps even giant cell formation in these tumors. Additional studies are required to confirm the role of p21 in the pathogenesis of these tumors.

Bone Neoplasms↗

PubMed2000

Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation.

GSK-3beta-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3beta could inhibit nuclear import. Here, we report that GSK-3beta-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3beta, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division.

Active Transport, Cell Nucleus↗

PubMed2000

PERK mediates cell-cycle exit during the mammalian unfolded protein response.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR)-signaling pathway. The UPR coordinates the induction of ER chaperones with decreased protein synthesis and growth arrest in the G(1) phase of the cell cycle. Three ER transmembrane protein kinases (Ire1alpha, Ire1beta, and PERK) have been implicated as proximal effectors of the mammalian UPR. We now demonstrate that activation of PERK signals the loss of cyclin D1 during the UPR, culminating in cell-cycle arrest. Overexpression of wild-type PERK inhibited cyclin D1 synthesis in the absence of ER stress, thereby inducing a G(1) phase arrest. PERK expression was associated with increased phosphorylation of the translation elongation initiation factor 2alpha (eIF2alpha), an event previously shown to block cyclin D1 translation. Conversely, a truncated form of PERK lacking its kinase domain acted as a dominant negative when overexpressed in cells, attenuating both cyclin D1 loss and cell-cycle arrest during the UPR without compromising induction of ER chaperones. These data demonstrate that PERK serves as a critical effector of UPR-induced growth arrest, linking stress in the ER to control of cell-cycle progression.

3T3 Cells↗

PubMed2000

Wnt1 and MEK1 cooperate to promote cyclin D1 accumulation and cellular transformation.

Members of the Wnt family of signal transducers regulate cellular differentiation/reorganization and cellular proliferation. However, few pro-proliferative targets of Wnt have been identified. We now show that cyclin D1, a critical mediator of cell cycle progression, is a downstream target of Wnt-dependent signaling. NIH-3T3 cell lines engineered to overexpress Wnt1 displayed reduced glycogen synthase kinase-3beta activity. Wnt1-dependent glycogen synthase kinase-3beta inhibition corresponded with decreased cyclin D1 proteolysis and, thus, hyperaccumulation of active cyclin D1.CDK4 (cyclin-dependent kinase 4) kinase. However, in the absence of serum-derived growth factors, Wnt-1 was not sufficient to drive cyclin D1 accumulation or S-phase entry. In contrast, cells engineered to co-express Wnt1 and activated MEK1 accumulated high levels of cyclin D1 and entered the DNA synthetic phase in the absence of serum-derived growth factors, a characteristic of neoplastic transformation. The ability of a dominant-negative cyclin D1 mutant, D1-T156A, to inhibit Wnt1/MEK1-dependent S-phase entry suggests that cyclin D1 is a critical downstream target for Wnt1- and MEK1-dependent cellular proliferation.

3T3 Cells↗

PubMed2000

Intention to be tested for prostate cancer risk among African-American men.

This study was conducted to identify factors associated with intention to be tested for prostate cancer risk among African-American men. Participants in this study included African-American men (n = 548) who were patients at the University Health Service at the University of Chicago, were 40 to 70 years of age, and did not have a personal history of prostate cancer. Baseline telephone survey data were collected for 413 (75%) men. Respondents were asked if they intended to have a blood test to assess prostate cancer risk. Univariate and multivariate analyses of intention to be tested for risk were performed. Eighty-six percent of the men said that they intended to be tested. Multivariate analysis results show that belief in the efficacy of prostate cancer screening [odds ratio (OR) = 3.6; 95% confidence interval (CI) = 1.4, 9.1] and intention to undergo a prostate cancer-screening (i.e., digital rectal examination and prostate-specific antigen testing; OR = 2.8; 95% CI = 1.3, 6.3) were positively associated with intention to be tested for prostate cancer risk. Being older (OR = 0.4; 95% CI = 0.2, 0.9), having had a prostate cancer-screening examination in the past year (OR = 0.5; 95% CI = 0.2, 1.0), perceiving one's prostate cancer susceptibility to be high (OR = 0.4; 95% CI = 0.2, 0.8), and being fatalistic about prostate cancer prevention (OR = 0.3; 95% CI = 0.2, 0.7) were negatively associated with intention to be tested for risk. Intention to be tested for prostate cancer risk was high among men in the study. Past screening, perceived susceptibility, and beliefs related to early detection might influence receptivity to genetic testing for prostate cancer risk.

Adult↗

PubMed1999

Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression.

Alterations in normal protein biogenesis and the resulting accumulation of improperly folded proteins in the endoplasmic reticulum (ER) trigger a stress response that up-regulates the expression of ER chaperones, while coordinately repressing overall protein synthesis and causing cell-cycle arrest. Activation of this unfolded protein response (UPR) in mouse NIH 3T3 fibroblasts with the glycosylation inhibitor tunicamycin led to a decline in cyclin D- and E-dependent kinase activities and to G(1) phase arrest. Cyclin D1 protein synthesis was rapidly inhibited by tunicamycin treatment. However, the drug did not significantly affect the mitogen-dependent activities of the extracellular signal-activated protein kinases ERK1 and ERK2 or the level of cyclin D1 mRNA until much later in the response. Therefore, the UPR triggers a signaling pathway that blocks cyclin D1 translation despite continuous mitogenic stimulation. Enforced overexpression of cyclin D1 in tunicamycin-treated cells maintained cyclin D- and E-dependent kinase activities and kept cells in cycle in the face of a fully activated UPR. Translational regulation of cyclin D1 in response to ER stress is a mechanism for checkpoint control that prevents cell-cycle progression until homeostasis is restored.

3T3 Cells↗

PubMed1999

Adherence by African American men to prostate cancer education and early detection.

BACKGROUND: This study was designed to identify factors that predict adherence by African American men to prostate cancer education and early detection. METHODS: In the spring of 1995, the authors identified 548 African American men who were patients at the University Health Services of the University of Chicago, were ages 40-70 years, and did not have a personal history of prostate cancer. Baseline telephone survey data were collected from 413 men (75%). Participants were randomly assigned to either a minimal or an enhanced intervention group. Men in the former group were mailed a letter and a reminder that invited them to a urology clinic for prostate cancer education and early detection. Men in the enhanced intervention group were sent the same correspondence and were also given print material and telephone contacts, which were tailored to each recipient. RESULTS: Adherence was significantly higher (OR = 2.6, CI: 1.7-3.9) in the enhanced intervention group than in the minimal intervention group (51% and 29%, respectively). Men who were age 50 years or older (OR = 1.7, CI: 1.1-2.8), were married (OR = 1.8, CI: 1.2-2.9), believed that prostate cancer early detection examination should be performed in the absence of symptoms (OR = 2.3, CI: 1.3-4.0), and self-reported an intention to have an early detection examination (OR = 1.9, CI: 1.2-2.9) were also more likely to adhere. CONCLUSIONS: A tailored behavioral intervention can influence adherence to prostate cancer early detection among African American men. Individual background and cognitive and psychosocial characteristics may also affect behavior. Future studies should assess the impact of this type of intervention on cognitive and psychologic correlates of decision-making and behavior along the continuum of prostate cancer care. [See editorial on pages 1-2, this issue.]

Adult↗

PubMed1999

Loss of the ARF tumor suppressor reverses premature replicative arrest but not radiation hypersensitivity arising from disabled atm function.

The alternative reading frame product (p19ARF) of the mouse INK4a/ARF locus is induced by oncoproteins such as Myc and E1A as part of a checkpoint response that limits cell cycle progression in response to hyperproliferative signals. ARF binds directly to Mdm2 to prevent down-regulation of p53 and thereby promotes p53-dependent transcription and cell cycle arrest. However, ARF is not required for p53 induction in response to ionizing radiation or other forms of DNA damage. Animals lacking a functional ataxia telangiectasia (Atm) gene are exquisitely sensitive to ionizing radiation; Atm-null mouse embryo fibroblasts (MEFs) undergo premature replicative arrest, which is relieved by the loss of p53. Here we show that the loss of ARF expands the life expectancy of Atm-null MEFs, but alters neither the sensitivity of Atm-null mice to ionizing radiation nor their propensity to develop lymphomas early in life. Therefore, whereas ARF and Atm signal to p53 through distinct pathways, the loss of ARF can modify p53-dependent features of the Atm-null phenotype.

Animals↗

PubMed1999

The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts.

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.

Animals↗

PubMed1999

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.

3T3 Cells↗

PubMed1999

Involvement of lipid mediators on cytokine signaling and induction of secretory phospholipase A2 in immortalized astrocytes (DITNC).

Our previous studies demonstrated the ability of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta), to stimulate NFkappaB/DNA binding and synthesis of secretory phospholipase A2 (sPLA2) in immortalized astrocytes (DITNC). In this study, we examined possible involvement of lipid mediators in the cytokine action. Using [14C]serine to label sphingomyelin and ceramide in these cells, subsequent exposure of cells to cytokines did not result in alteration of sphingomyelin/ceramide ratio. Furthermore, neither exogenous sphingomyelinase nor cell-permeable ceramides could stimulate NFkappaB/DNA binding. On the other hand, C-2 ceramide (0.3 microM) as well as other lipid mediators, such as lysophosphatidylcholine and arachidonic acid, were able to elicit a small increase in sPLA2 and potentiate the induction of sPLA2 by TNF-alpha. When DITNC cells were prelabeled with [32P]Pi, an increase in labeled phosphatidic acid (PA) was observed on treatment of cells with IL-1beta (200 U/mL). However, despite the ability of phorbol myristate acetate (PMA) to stimulate phospholipase D (PLD) and synthesis of phosphatidylethanol (PEt) in these cells, PLD activity was not affected by IL-1beta. With the [32P]labeled cells, however, PA-phosphohydrolase inhibitors, such as chlorpromazine and propranolol, could elicit large increases in labeled PA, indicating active PA metabolism in these cells. Cytokines also caused an increase in levels of diacylglycerol (DG) in these cells, although the source of this lipid pool is presently not understood. Taken together, these results provide evidence for the participation of PA and DG in cytokine signaling activity. Furthermore, although cytokines did not cause the release of ceramide, lipid mediators, such as lysophospholipids, and AA could modulate cytokine-mediated induction of sPLA2 in astrocytes.

Animals↗

PubMed1998

Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization.

The activities of cyclin D-dependent kinases serve to integrate extracellular signaling during G1 phase with the cell-cycle engine that regulates DNA replication and mitosis. Induction of D-type cyclins and their assembly into holoenzyme complexes depend on mitogen stimulation. Conversely, the fact that D-type cyclins are labile proteins guarantees that the subunit pool shrinks rapidly when cells are deprived of mitogens. Phosphorylation of cyclin D1 on a single threonine residue near the carboxyl terminus (Thr-286) positively regulates proteasomal degradation of D1. Now, we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) phosphorylates cyclin D1 specifically on Thr-286, thereby triggering rapid cyclin D1 turnover. Because the activity of GSK-3beta can be inhibited by signaling through a pathway that sequentially involves Ras, phosphatidylinositol-3-OH kinase (PI3K), and protein kinase B (Akt), the turnover of cyclin D1, like its assembly, is also Ras dependent and, hence, mitogen regulated. In contrast, Ras mutants defective in PI3K signaling, or constitutively active mitogen-activated protein kinase-kinase (MEK1) mutants that act downstream of Ras to activate extracellular signal-regulated protein kinases (ERKs), cannot stabilize cyclin D1. In direct contrast to cyclin D1, which accumulates in the nucleus during G1 phase and exits into the cytoplasm during S phase, GSK-3beta is predominantly cytoplasmic during G1 phase, but a significant fraction enters the nucleus during S phase. A highly stable D1 mutant in which an alanine is substituted for the threonine at position 286 and that is refractory to phosphorylation by GSK-3beta remained in the nucleus throughout the cell cycle. Overexpression of an active, but not a kinase-defective, form of GSK-3beta in mouse fibroblasts caused a redistribution of cyclin D1 from the cell nucleus to the cytoplasm. Therefore, phosphorylation and proteolytic turnover of cyclin D1 and its subcellular localization during the cell division cycle are linked through the action of GSK-3beta.

3T3 Cells↗

PubMed1997

A threshold nuclear level of the v-Rel oncoprotein is required for transformation of avian lymphocytes.

The net distribution of eukaryotic transcription factors between the cytoplasm and the nucleus provides an effective mechanism for controlling gene expression. We have utilized cis-acting signals for both nuclear import and nuclear export to experimentally manipulate the distribution of the v-Rel oncoprotein between the nucleus and the cytoplasm. The respective abilities of the v-Rel oncoprotein to localize to the nucleus in chicken embryo fibroblasts, to activate kappaB-dependent transcription in yeast, and to transform avian lymphoid cells were each markedly reduced by the fusion of a cis-acting nuclear export signal onto v-Rel. Our results demonstrate that a threshold nuclear function of v-Rel is required for manifestation of its oncogenic properties. In contrast, while increased expression of the avian IkappaB-alpha protein was able to prevent nuclear localization of v-Rel in chicken embryo fibroblasts, coexpression of IkappaB-alpha with v-Rel in the target cell for v-Rel mediated transformation did not reduce the ability of v-Rel to transform avian lymphoid cells or alter the distribution of v-Rel between the nucleus and the cytoplasm in v-Rel-transformed cells. Our results suggest that the ability of IkappaB-alpha to inhibit nuclear localization of v-Rel is affected by cell-type specific differences between fibroblasts and lymphoid cells.

Animals↗

PubMed1997

Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway.

The expression of D-type G1 cyclins and their assembly with their catalytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induced signals. In turn, the ability of cyclin D-dependent kinases to trigger phosphorylation of the retinoblastoma (Rb) protein in the mid- to late G1 phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth factors. However, the mechanisms governing the turnover of D-type cyclins have not yet been elucidated. We now show that cyclin D1 turnover is governed by ubiquitination and proteasomal degradation, which are positively regulated by cyclin D1 phosphorylation on threonine-286. Although "free" or CDK4-bound cyclin D1 molecules are intrinsically unstable (t1/2 < 30 min), a cyclin D1 mutant (T286A) containing an alanine for threonine-286 substitution fails to undergo efficient polyubiquitination in an in vitro system or in vivo, and it is markedly stabilized (t1/2 approximately 3.5 hr) when inducibly expressed in either quiescent or proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on threonine-286 also occurs in insect Sf9 cells, and although the process is enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinase can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated.

3T3 Cells↗

PubMed1997

A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

3T3 Cells↗