Fascin-1 expression is associated with neuroendocrine prostate cancer and directly suppressed by androgen receptor.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is an aggressive form of prostate cancer, arising from resistance to androgen-deprivation therapies. However, the molecular mechanisms associated with NEPC development and invasiveness are still poorly understood. Here we investigated the expression and functional significance of Fascin-1 (FSCN1), a pro-metastasis actin-bundling protein associated with poor prognosis of several cancers, in neuroendocrine differentiation of prostate cancer. METHODS: Differential expression analyses using Genome Expression Omnibus (GEO) database, clinical samples and cell lines were performed. Androgen or antagonist's cellular treatments and knockdown experiments were used to detect changes in cell morphology, molecular markers, migration properties and in vivo tumour growth. Chromatin immunoprecipitation-sequencing (ChIP-Seq) data and ChIP assays were analysed to decipher androgen receptor (AR) binding. RESULTS: We demonstrated that FSCN1 is upregulated during neuroendocrine differentiation of prostate cancer in vitro, leading to phenotypic changes and NEPC marker expression. In human prostate cancer samples, FSCN1 expression is restricted to NEPC tumours. We showed that the androgen-activated AR downregulates FSCN1 expression and works as a transcriptional repressor to directly suppress FSCN1 expression. AR antagonists alleviate this repression. In addition, FSCN1 silencing further impairs in vivo tumour growth. CONCLUSION: Collectively, our findings identify FSCN1 as an AR-repressed gene. Particularly, it is involved in NEPC aggressiveness. Our results provide the rationale for the future clinical development of FSCN1 inhibitors in NEPC patients.

Aberrant up-regulation of iNOS/NO system is correlated with an increased abundance of Foxp3+ cells and reduced effector/memory cell markers expression during colorectal cancer: immunomodulatory effects of cetuximab combined with chemotherapy.

Colorectal cancer (CRC) remains the most cancer type related to chronic inflammation; however, the mechanisms that link inflammation to CRC development and progression are still poorly understood. Our study aimed to investigate one of the prominent inflammatory response in cancers, iNOS/NO system. In this regard, we evaluated the link between the iNOS/NO system and CRC progression, its relation with the host immune responses and its response to cetuximab combined with chemotherapy. We found that the nitrite levels were nearly twice as high in metastatic CRC plasma and culture supernatants from PBMCs and tumor explants compared with those without metastases and healthy controls. Interestingly, we showed that the highest iNOS expression and NO levels are present in the damaged CRC tissues that have highest leukocyte infiltration. Our findings highlight the implication of iNOS/NO system in tissue alteration and leukocyte invasion. Thus, we observed imbalance between effector/memory T cell markers and Treg transcription factor (Foxp3). Accordingly, we detected higher IFNγ and T-bet expression levels in colorectal tumor tissues at early stage. In contrast, consistent with iNOS and Foxp3 expression, TGFß, CTLA-4 and IL-10 were significantly related to the tumor stage progression. Furthermore, our study revealed that Cetuximab combined with chemotherapy treatment markedly down-regulates iNOS/NO system as well as IL-10 and TGFß levels. Altogether, we conclude that cetuximab can potentiate the efficacy of chemotherapy, particularly by iNOS/NO system and immunosuppressive cytokines modulation. Thus, we suggest that iNOS/NO system may represent an attractive candidate biomarker for monitoring CRC progression, malignity and response to therapy.

ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis.

BACKGROUND: The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis. METHODS: Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples. RESULTS: Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer. CONCLUSION: MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.

All-Trans Retinoic Acid Modulates TLR4/NF-κB Signaling Pathway Targeting TNF-α and Nitric Oxide Synthase 2 Expression in Colonic Mucosa during Ulcerative Colitis and Colitis Associated Cancer.

Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF-κB signaling in development and progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF-κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF-α, TLR4, and NF-κB, in colonic mucosa. We also studied NO/TNF-α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF-κB, TNF-α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF-α/NO production, as well as the role of NF-κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF-α induction through NF-κB pathway was suggested. AtRA downregulates NOS2 and TNF-α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF-κB signaling pathway targeting NOS2 and TNF-α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.

The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members.

PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro. Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.

Regulation of ploidy and senescence by the AMPK-related kinase NUAK1.

Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.

Epstein-Barr virus infection induces an increase of T regulatory type 1 cells in Hodgkin lymphoma patients.

Epstein-Barr Virus (EBV) is present in the neoplastic cells of around 20-30% of patients with Hodgkin Lymphoma (HL). Although, an immunosuppressive environment is currently described in HL patients, little is known concerning the regulatory mechanism induced by EBV proteins expression in tumour cells. This study aimed to investigate an association between regulatory Type 1 cells (Tr1) and EBV tissue positivity in HL patients. Transcriptomic analysis of both EBV-positive and EBV-negative tumours showed that EBV infection increased gene expression of Tr1-related markers (ITGA2, ITGB2, LAG3) and associated-immunosuppressive cytokines (IL10). This up-regulation was associated with an over-expression of several chemokine markers known to attract T-helper type 2 (Th2) and regulatory T cells thus contributing to immune suppression. This Tr1 cells recruitment in EBV-positive HL was confirmed by immunohistochemical analysis of frozen nodes biopsies and by flow cytometric analysis of peripheral blood mononuclear cells of EBV-positive patients. Additionally, we showed that IL10 production was significantly enhanced in tumours and blood of EBV-positive HL patients. Our results propose a new model in which EBV can recruit Tr1 cells to the nodes' microenvironment, suggesting that the expression of EBV proteins in tumour cells could enable the escape of EBV-infected tumour cells from the virus-specific CTL response.

[Sorting of senescent cells by flow cytometry: Specificities and pitfalls to avoid]. / Tri des cellules sénescentes par cytométrie en flux - Des spécificitéset des pièges à éviter.

Cells can be reprogrammed into senescence to adapt to a variety of stresses, most often affecting the genome integrity. Senescent cells accumulate with age or upon various insults in almost all tissues, and contribute to the development of several age-associated pathologies. Studying the molecular pathways involved in senescence induction, maintenance, or escape is challenged by the heterogeneity in the level of commitment to senescence, and by the pollution of senescent cell populations by proliferating pre- or post-senescent cells. We coped with these difficulties by developing a protocol for sorting senescent cells by flow cytometry, based on three major senescence markers : the SA-ß-Galactosidase activity, the size of the cells, and their granularity reflecting the accumulation of aggregates, lysosomes, and altered mitochondria. We address the issues related to sorting senescent cells, the pitfalls to avoid, and propose solutions for sorting viable cells expressing senescent markers at different extents.

Title: Tri des cellules sénescentes par cytométrie en flux - Des spécificitéset des pièges à éviter. Abstract: La sénescence est un état d'adaptation des cellules au stress qui contribue au vieillissement et au développement de nombreuses maladies. Étudier les voies moléculaires modulant l'induction, le maintien ou l'échappement de la sénescence est compliqué par la contamination des populations de cellules sénescentes par des cellules proliférantes pré- ou post-sénescentes. Pour contourner cette difficulté, nous avons développé un protocole de tri par cytométrie en flux, fondé sur trois marqueurs majeurs de sénescence (l'activité SA-ß-galactosidase, la taille et la granularité des cellules), qui permet de trier des cellules sénescentes viables, à des degrés choisis d'engagement dans le phénotype.

Anti-apoptotic role of caspase-cleaved GAB1 adaptor protein in hepatocyte growth factor/scatter factor-MET receptor protein signaling.

The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling.

Flow Cytometry-based Method for Efficient Sorting of Senescent Cells.

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-ß-galactosidase (SA-ß-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-ß-D-galactopyranoside (C12FDG), a fluorigenic substrate of ß-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents. Graphical abstract.

The Polycomb group protein EZH2 directly controls DNA methylation.

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.

The coactivator activator CoAA regulates PEA3 group member transcriptional activity.

The PEA3 (polyoma enhancer activator 3) group members [ERM (ETS-related molecule), ER81 (ETS-related 81) and PEA3] of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the hnRNP (heterogeneous nuclear ribonucleoprotein)-like protein CoAA (Coactivator activator) interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RRMs (RNA recognition motifs) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group member activity during metastasis.

NMR structure of the human Mediator MED25 ACID domain.

MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed ß-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the ß-barrel, defined by strands B4-B7-B6.

Purification of SUMO-1 modified IκBα and complex formation with NF-κB.

Covalent modification of proteins with SUMO (Small Ubiquitin-like MOdifier) affects many cellular processes, including transcriptional regulation, DNA repair and signal transduction. Although hundreds of SUMO targets have been identified, many biological outcomes of protein sumoylation remain poorly understood. In particular, biochemical and structural analysis can only be easily conducted if highly pure sumoylated substrates are available. Purification of sumoylated substrates in vitro or in bacteria have been previously reported but separating the sumoylated protein from the undesired unmodified fraction is often technically challenging, inefficient and time consuming. Here we develop a new vector system for in vivo sumoylation in Escherichia coli which improves purification of sumoylated proteins. We describe the purification of IκBα, its sumoylation, the subsequent separation and purification of the modified and the unmodified forms and the purification of the complex IκBα-SUMO-1/NF-κB. After a first GST affinity chromatography and GST-tag removal, a unique metal-ion affinity chromatography using a 6xHis-SUMO-1 tag results in mgs of highly pure SUMO-1 modified IκBα. Our pure SUMO-1 modified IκB/NF-κB complex could be a useful tool to identify new interaction partner specific of the SUMO-1 modified IκBα form. This approach may be extended to other SUMO substrates not isolable by classical chromatography techniques.

The M-type receptor PLA2R regulates senescence through the p53 pathway.

Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss-of-function genetic screen in primary human fibroblasts. We report that knockdown of the M-type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress-induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species-DNA damage-p53-dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

Senescent keratinocytes die by autophagic programmed cell death.

Normal cells reach senescence after a specific time and number of divisions, leading ultimately to cell death. Although escape from this fate may be a requisite step in neoplastic transformation, the mechanisms governing senescent cell death have not been well investigated. We show here, using normal human epidermal keratinocytes, that no apoptotic markers appear with senescence. In contrast, the expression of several proteins involved in the regulation of macroautophagy, notably Beclin-1 and Bcl-2, was found to change with senescence. The corpses occurring at the senescence growth plateau displayed a large central area delimited by the cytokeratin network that contained a huge quantity of autophagic vacuoles, the damaged nucleus, and most mitochondria. 3-methyladenine, an inhibitor of autophagosome formation, but not the caspase inhibitor zVAD, prevented senescent cell death. We conclude that senescent cells do not die by apoptosis, but as a result of high macroautophagic activity that targets the primary vital cell components.

ERM transcription factor contains an inhibitory domain which functions in sumoylation-dependent manner.

ERM, PEA3 and ETV1 belong to the PEA3 group of ETS transcription factors. They are involved in many developmental processes and are transcriptional regulators in metastasis. The PEA3 group members share an N-terminal transactivation domain (TAD) whose activity is inhibited by a flanking domain named the negative regulatory domain (NRD). The mechanism of this inhibition is still unknown. Here we show that the NRD maps to residues 73 to 298 in ERM and contains three of the five SUMO sites previously identified in the protein. We demonstrate that these three SUMO sites are responsible for NRD's inhibitory function in the Gal4 system. Although the presence of the three sites is required to obtain maximal inhibition, only one SUMO site is sufficient to repress transcription whatever its localization within the NRD. We also show that NRD is a SUMO-dependent repression domain that can act in cis and in trans to downregulate the powerful TAD of the VP16 viral protein. In addition, we find that the SUMO sites outside the NRD also play a role in the negative regulation of full-length ERM activity. We thus postulate that each SUMO site in ERM may function as an inhibitory motif.

Functional cooperation between Stat-1 and ets-1 to optimize icam-1 gene transcription.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the immune system, enabling the interactions between effector cells and target cells. It is also known to be involved in tumor growth and metastasis. Its expression is transcriptionally regulated by several proinflammatory cytokines including IFN-gamma, which induces ICAM-1 transcription via the JAK-STAT signaling pathway in a Stat1-dependent fashion. The ICAM-1 promoter contains several cis-active regulatory elements including 2 Ets binding sites (EBSs) located at positions -158 and -138 relatively to the AUG, which were previously shown to play a role in the constitutive activity of the ICAM-1 promoter. In the present study, we have determined whether the EBSs are also involved in the regulation of ICAM-1 gene transcription by pro-inflammatory cytokines. Transient transfection assays were performed with reporter genes containing ICAM-1 promoter constructions cloned upstream from the firefly luciferase gene. Site-specific mutations of the EBS diminished the promoter activity stimulated by IFN-gamma, although the IFN-gamma responsive element (pIgammaRE), which binds Stat1, was intact. Stimulation of the transcriptional activity following IFN-gamma treatment was significantly reduced when both EBSs were inactivated. Co-immunoprecipitation experiments provided evidence of a physical interaction involving Ets1 and Stat1. In COS-1 and HEK 293 cells cotransfected with CFP-Stat1 and YFP-Ets fusion protein, fluorescence resonance energy transfer experiments confirmed the close proximity of these 2 proteins in living cells following treatment with IFN-gamma.

Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors".

Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.