Laurence Bockstaele

Rb inactivation in cell cycle and cancer: the puzzle of highly regulated activating phosphorylation of CDK4 versus constitutively active CDK-activating kinase.

Cyclin-dependent kinase (CDK) 4 is a master integrator that couples mitogenic/oncogenic signalling cascades with the inactivation of the central oncosuppressor Rb and the cell cycle. Its activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 by the only identified CDK-activating kinase in animal cells, cyclin H-CDK7. In contrast with the observed constitutive activity of cyclin H-CDK7, we have recently identified the T172-phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Intriguingly, the homologous T177-phosphorylation of CDK6 is weak in several systems and does not present this regulation. In this Perspective, we review the recent advances and debates on the multistep mechanism leading to activation of D-type cyclin–CDK4 complexes. This involves a re-evaluation of the implication of Cip/Kip CDK “inhibitors” and CDK7 in this process.

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

To control the G1/S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27kip1 and p21cip1) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15- or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.

Regulated Activating Thr172 Phosphorylation of Cyclin-Dependent Kinase 4(CDK4): Its Relationship with Cyclins and CDK "Inhibitors"

ABSTRACT 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.

Differential regulation of cyclin-dependent kinase 4 (CDK4) and CDK6, evidence that CDK4 might not be activated by CDK7, and design of a CDK6 activating mutation.

ABSTRACT The homologous cyclin-dependent kinases (CDK) CDK4 and CDK6 integrate mitogenic and oncogenic signaling cascades with the cell cycle. Their activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 and T177 (respectively) by the only CDK-activating kinase identified in animal cells, cyclin H-CDK7. At odds with the existing data showing the constitutive activity of CDK7, we have recently identified the T172 phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Here we show that T172 phosphorylation of CDK4 is conditioned by its unique proline 173 residue. In contrast to CDK4, CDK6 does not contain such a proline and, unexpectedly, remained poorly phosphorylated and active in a variety of cells. Mutations of proline 173 did not adversely affect CDK4 activation by CDK7, but in cells they abolished CDK4 T172 phosphorylation and activity. Conversely, substituting a proline for the corresponding residue of CDK6 enforced its complete, apparently cyclin-independent T177 phosphorylation and dramatically increased its activity. These results lead us to propose that CDK4 might not be phosphorylated by CDK7 in intact cells but is more likely phosphorylated by another, presumably proline-directed kinase(s). Moreover, they provide a new model of a potentially oncogenic activating mutation of a CDK.

The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFβ, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis

The cAMP-dependent mitogenic stimulation elicited by thyroid-stimulating hormone (TSH) in primary cultures of canine thyroid epithelial cells is unique as it upregulates the cyclin-dependent kinase (CDK) inhibitor p27kip1 but not D-type cyclins. TSH and cAMP promote the assembly of required cyclin D3-CDK4 complexes and their nuclear import. Here, the nuclear translocation of these complexes strictly correlated in individual cells with the enhanced presence of nuclear p27. p27, like cyclin D3, supported the TSH-stimulated pRb-kinase activity of the CDK4 complex and, as demonstrated using the high-resolution power of the two-dimensional (2D) gel electrophoresis, the phosphorylation of CDK4, presumably by the nuclear CDK-activating kinase. In the presence of TSH, transforming growth factor beta (TGFbeta) did not affect the assembly of cyclin D3-CDK4, but it strongly inhibited the pRb-kinase activity associated with both cyclin D3 and p27, not only by preventing the nuclear import of cyclin D3-CDK4 and its binding to p27, but also by inhibiting CDK4 phosphorylation within residual p27-bound cyclin D3-CDK4 complexes. No alterations of the relative abundance of multiple (un)phosphorylated forms of cyclin D3 and p27 demonstrated by 2D-gel electrophoresis were associated with these processes. This study suggests a crucial positive role of p27 in the TSH-stimulated nuclear import, phosphorylation, and catalytic activity of cyclin D3-bound CDK4. Moreover, it demonstrates a technique to directly assess the in vivo phosphorylation of endogenous CDK4, which might appear as a last regulated step targeted by the antagonistic cell cycle effects of TSH and TGFbeta.

Distinct Specificities of pRb Phosphorylation by CDK4 Activated by Cyclin D1 or Cyclin D3: Differential Involvement in the Distinct Mitogenic Modes of Thyroid Epithelial Cells

Two distinct mitogenic modes coexist in the physiologically relevant model ofprimary cultures of dog thyroid epithelial cells. The differentiation-associated mitogenicstimulation by TSH and cAMP specifically requires the assembly and activation of cyclin D3-cyclin-dependent kinase (CDK)4 associated to p27kip1, while the dedifferentiatingproliferation induced by growth factors is associated with induction of cyclin D1. Here, wesuggest that the related CDK “inhibitors” p21cip1 and p27 are differentially utilized as positiveCDK4 regulators in these mitogenic stimulations. p21 was induced by EGF+serum, butrepressed by TSH, which, as previously shown, up-regulates p27. In response to EGF+serum,p21 supported the nuclear localization, phosphorylation and pRb-kinase activity of CDK4.Unexpectedly, partly different site-specificities of pRb-kinase activity, leading to similardifferences in the phosphorylation pattern of pRb in intact cells, were associated with cyclinD3-CDK4 bound to p27 in TSH-stimulated cells, or with CDK4 bound to p21 in growthfactor-stimulated cells. These differences were ascribed to the predominant association of thelatter complex to cyclin D1. Indeed, in different cell types and species, cyclin D1 varied fromcyclin D3 by more efficiently driving the phosphorylation of pRb at sites (Ser807/811 andThr826) required for its electrophoretic mobility shift. Therefore, different D-type cyclinscould differently impact some pRb functions, which should be considered not only in theunderstanding of the relationships between cell cycle and differentiation expression in thedistinct mitogenic modes of thyroid cells, but also in various development or differentiationmodels associated with dramatic switches in the expression of individual D-type cyclins.

Safety of Ovarian Tissue Autotransplantation for Cancer Patients

Cancer treatments can induce premature ovarian failure in almost half of young women suffering from invasive neoplasia. Cryopreservation of ovarian cortex and subsequent autotransplantation of frozen-thawed tissue have emerged as promising alternatives to conventional fertility preservation technologies. However, human ovarian tissue is generally harvested before the administration of gonadotoxic treatment and could be contaminated with malignant cells. The safety of autotransplantation of ovarian cortex remains a major concern for fertility preservation units worldwide. This paper discusses the main tools for detecting disseminated cancer cells currently available, their limitations, and clinical relevance.

Evaluation of quantitative polymerase chain reaction markers for the detection of breast cancer cells in ovarian tissue stored for fertility preservation

OBJECTIVE To develop molecular tools increasing the sensitivity of breast cancer micrometastases detection within ovarian tissue cryopreserved for fertility preservation. DESIGN Expression of breast markers was evaluated by quantitative polymerase chain reaction in ovarian tissue from patients with benign or cancerous diseases. Suspected tissues were long-term xenografted into mice. SETTING Academic research institute. PATIENT(S) Patients undergoing a fertility preservation procedure. INTERVENTION(S) Ovarian tissue was processed for RNA extraction and quantitative polymerase chain reaction analysis. Cryopreserved ovarian cortex from patients with breast cancer or benign disease was grafted for 6 months into severe combined immunodeficiency mice. MAIN OUTCOMES MEASURE(S) Predictive values of mammaglobin 1 (MGB-1), gross cystic disease fluid protein-15 (GCDFP-15), small breast epithelial mucine (SBEM), and mammaglobin 2 (MGB-2) to detect breast cancer cells in ovarian tissue, and the potential development of cancerous disease after xenograft of ovarian cortex from breast cancer patients. RESULT(S) MGB-1 and GCDFP-15 presented the highest predictive values to detect breast cancer micrometastases in the ovarian cortex, with an efficiency reaching 100% and 77%, respectively. The MGB-2 assay resulted in a high false-positive rate (47%) in the ovarian cortex but could be used to detect breast cancer cells in ovarian medulla. MGB-1 was detected in three of five ovarian cortex samples from early-stage breast cancer patients but not in the ovarian tissue from advanced breast cancer patients (none of 10). None of the mice grafted with ovarian tissue expressing these markers developed cancerous disease. CONCLUSION(S) MGB-1, GCDFP-15, and MGB-2 can serve as molecular markers for the detection of breast cancer micrometastases within the ovarian tissue of breast cancer patients. However, the clinical relevance of such a highly sensitive assay must be further investigated.