Martha J. Grossel

From cell cycle to differentiation: an expanding role for cdk6.

Over ten years ago, cdk6 was identified as a new member in a family of vertebrate cdc-2 related kinases. This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro. 1 Recently, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. Since exit from the cell cycle is a necessary step in the process of differentiation, it may not seem surprising that down-regulation of a mitogenic factor may be required for this process. It is, however, surprising that this association has not been previously uncovered and that it is apparently not a shared with cdk4, long understood to be a functional homolog of cdk6. As this story unfolds it will be important to discover if the role of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation.

Beyond the cell cycle : A new role for Cdk6 in differentiation

Over 10 years ago, cdk6 was identified as a new member in a family of vertebrate cdc‐2 related kinases. This novel kinase was found to partner with the D‐type cyclins and to possess pRb kinase activity in vitro and has since been understood to function solely as a pRb kinase in the regulation of the G1 phase of the cell cycle. In the past 2 years, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. For example, cdk6 expression must be reduced to allow proper osteoblast and osteoclast differentiation, forced cdk6 expression blocked differentiation of mouse erythroid leukemia cells and cdk6 expression in primary astrocytes favors the expression of progenitor cell markers. Since exit from the cell cycle is a necessary step in terminal differentiation, down‐regulation of a mitogenic factor may be expected in this process, however it is surprising that this association has not been previously uncovered and that it is apparently not shared with cdk4, long understood to be a functional homolog of cdk6. The mechanism of cdk6 function in differentiation is not understood, but it may extend beyond the established role of cdk6 as a pRb kinase. As this story unfolds it will be important to discover if the function of cdk6 in differentiation is pRb‐dependent or pRb‐independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation.

Science in the News: A Study of Reporting Genomics

Scientists and academicians in the field of science writing agree that context and method are important components of research to be reported. This ideal is balanced by science journalists who try to relay findings with a minimum of complex and potentially confusing facts. Here, a specific report on genomics is traced from its original source in a scientific journal through to popular press publications. These data were examined in the context of previously published findings that have shown that the reader needs a clear understanding of the context of reported results to make an informed judgment about their meaning. This study reveals that these five reports, ranging from research article to popular press news article, differ in what is said rather than how it is said. This is surprising given the premise that in science reporting, the primary role of the journalist is to translate science into non-scientific language.

Changes in motility, gene expression and actin dynamics: Cdk6‐induced cytoskeletal changes associated with differentiation in mouse astrocytes

Cyclin dependent kinase (cdk) 4 and cdk6 have historically been understood to be D‐cyclin kinases that phosphorylate pRb in the nucleus to regulate G1 phase of the cell cycle. In conflict with this understood redundancy are several studies that have demonstrated a novel role for cdk6 in differentiation. Cdk6 expression must be reduced to allow proper osteoblast and osteoclast differentiation, enforced cdk6 expression blocked differentiation of mouse embryo fibroblasts, and cdk6 expression in primary astrocytes favored the expression of progenitor cell markers (Ericson et al. [2003] Mol Cancer Res 1:654–664; Matushansky et al. [2003] Oncogene 22:4143–4149; Ogasawara et al. [2004a] J Bone Miner Res 19:1128–1136; Ogasawara et al. [2004b] Mol Cell Biol 24:6560–6568). Experiments shown here investigate novel cytoplasmic and nuclear functions of cdk6. These data demonstrate that cdk6 expression in mouse astrocytes results in changes in patterns of gene expression, changes in the actin cytoskeleton including loss of stress fibers, and enhanced motility. These changes in cdk6‐infected cells are associated with the process of cellular differentiation. J. Cell. Biochem. 99: 635–646, 2006.

Distinct subcellular distribution of cyclin dependent kinase 6

Several studies have recently reported that the cyclin dependent kinase (cdk) 6 oncogene plays a role in differentiation of a variety of cell types. This novel function expands the previously understood function of cdk6 as a regulator of G1 phase of the cell cycle. The proposed mechanisms of these functions both require nuclear localization. That is, cdk6 phosphorylation of the retinoblastoma protein (pRb) to regulate cell cycle, and the recently proposed transcriptional regulation to block differentiation, are both nuclear functions that predict nuclear localization of the kinase. This report provides a thorough analysis of cdk6 localization and compares the localization of a commonly used mutant cdk6 to the corrected wildtype sequence as recorded in GenBank. The widely shared mutant of cdk6 contains a tyrosine residue at amino acid 224 (instead of an aspartic acid) introducing a potential phosphorylation site to the cdk6 sequence. Results indicate a majority of cdk6 is localized to the cytoplasm with concentrations of cdk6 in the edges of the cytoplasm and in the cytoplasmic extensions of cells. The results of this study may help to better understand the emerging roles of cdk6 in cell cycle control, differentiation and cancer.

An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promegas luc2 for reporter and imaging applications.

CDK6 binds and promotes the degradation of the EYA2 protein

Cyclin-dependent kinase 6 (Cdk6) is a D-Cyclin-activated kinase that is directly involved in driving the cell cycle through inactivation of pRB in G1 phase. Increasingly, evidence suggests that CDK6, while directly driving the cell cycle, may only be essential for proliferation of specialized cell types, agreeing with the notion that CDK6 also plays an important role in differentiation. Here, evidence is presented that CDK6 binds to and promotes degradation of the EYA2 protein. The EYA proteins are a family of proteins that activate genes essential for the development of multiple organs, regulate cell proliferation, and are misregulated in several types of cancer. This interaction suggests that CDK6 regulates EYA2 activity, a mechanism that could be important in development and in cancer.

Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments

Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Km values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Km value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Km value in low ATP environments.

Abstract A38: In vitro and in vivo interaction of Cdk6 and Eya2 indicate potential crosstalk

We have identified a novel association of the developmentally significant protein, Eya2, with cyclin dependent kinase (cdk) 6. Eya2 is part of the conserved retinal determination network that has been shown to function in fly eye development (Rebay, 2005). In mammals, the EYA gene product has been implicated in gonadogenesis, myogenesis, neurognenesis, limb formation, thymus, and kidney development, in part through its ability to regulate cell proliferation (Zhang 2005). Eya2 is overexpressed in several types of cancers including epithelial ovarian (Zhang et al, 2005), cervical (Bierkens et al, 2013), lung adenocarcinoma (Guo et al, 2009), breast and hematopoietic (Patrick, 2013, Wang, 2011). Cdk6 knockout mice display defects in hematopoiesis, including decreased cellularity of the thymus, red blood cells and lymphocytes (Malumbres, 2004). Cdk6 kinase activity is required for thymocyte development (Hu et al, 2011) and this role is partially mediated by modulating Notch target gene expression. The interaction of these two proteins, each important in both development and cancer, and both involved in regulation of cell proliferation and transcription, was first identified in a yeast two-hybrid analysis of a human fetal brain library. Subsequent results from our labs confirm the association of cdk6 and Eya2 in GST binding assays and co-immunoprecipitations. GST binding assays demonstrated that Six4, a cofactor of Eya2, competes with cdk6 for Eya2 binding. Co-immunoprecipitation of transfected cell lysates and native immunoprecipitations also demonstrate binding. Finally, co-expression of cdk6 with Eya2 reduces the half-life of the Eya2 protein in cell lysates. We propose that cdk6 and Eya2 interact to affect the function of cell proliferation and differentiation that is crucial to both the biology of cancer and development. This interaction could provide a mechanism to allow the expansion of the progenitor cell population prior to differentiation. References: Rebay I, Silver SJ, Tootle TL (2005) Trends Genet 21: 163-171. Zhang L, et al. (2005). Cancer Research 65: 925-932. Bierkens M, et al. (2013). Genes, Chromosomes & Cancer 52: 56-68. Guo JT et al. (2009) Chinese journal of oncology 31: 528-531. Patrick AN, et al. (2013) Nature Structural & Molecular Biology 20: 447-453. Wang QF, et al. (2011) Blood 117: 6895-6905. Malumbres et al. (2004). Cell 118: 493-504. Hu MG, et al. (2011) Blood 117: 6120-6131. Citation Format: Dawn Kohrt, Philip Hinds, Heide Ford, Martha Grossel. In vitro and in vivo interaction of Cdk6 and Eya2 indicate potential crosstalk. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A38.

Abstract A18: CDK6 binds and promotes the degradation of the EYA2 protein

Cyclin dependent kinase 6 (Cdk6) is a D-cyclin activated kinase that is directly involved in driving the cell cycle through inactivation of pRB in G1 phase. Increasingly, evidence suggests that CDK6, while directly driving the cell cycle, may only be essential for proliferation of specialized cell types, agreeing with the notion that CDK6 also plays an important role in differentiation. Here, evidence is presented that CDK6 binds to and promotes degradation of the EYA2 protein. The EYA proteins are a family of proteins that activate genes essential for the development of multiple organs, regulate cell proliferation, and are misregulated in several types of cancer. This interaction suggests that CDK6 regulates EYA2 activity, a mechanism that could be important in development and in cancer. Citation Format: Dawn Kohrt, Jennifer Crary, Marc Zimmer, Heide Ford, Phi Hinds, Martha Grossel. CDK6 binds and promotes the degradation of the EYA2 protein. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A18.