Mammalian Cell Cycle Control Mechanisms Prof. Ingrid Hoffmann

Scientific Projects:

Mammalian Cell Cycle Control Mechanisms

Figure 1

Figure 1: The eukaryotic cell cycle is comprised of four phases: G1, S, G2 and M. Cyclin-dependent kinases are the key regulators of the cell cycle in eukaryotic cells

The cell cycle is an essential mechanism by which all living cells reproduce. A mammalian cell must tightly regulate each of its cell-cycle phase transitions to accurately transmit a copy of its genome to daughter cells. Eukaryotic cells have evolved a complex network of regulatory proteins known as the cell cycle control system, that governs progression through the cell cycle. Cyclin-dependent kinases, Cdks (Figure 1) regulate cell-cycle transitions and their activity is in turn controlled by various positive and negative upstream mechanisms.

Ongoing projects in the lab

Figure 2: siRNA-mediated down-regulation of Plk2 leads to the formation of monopolar spindles, immunostained with anti-α-tubulin (green) and centriole specific antibodies (GT335) (red), Scale bar, 10 µm

When cells divide, it is essential that they segregate their newly duplicated chromosomes into two equal sets. Missegregation not only distorts the number of chromosomes in the daughter cells but also elevates or diminishes the expression of genes critical for cell viability and growth. The equal segregation of sister chromatids at mitosis is directly linked to the organization of the microtubule-based mitotic spindle that guides chromosome separation.
Centrosomes are the microtubule-organizing centers (MTOCs) of animal cells. They contain a pair of barrel-shaped centrioles that are surrounded by pericentriolar material, PCM. Centrioles are cylindrical structures built of microtubules and closely related to basal bodies, which are essential for the formation of cilia and flagella. Centrioles duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. Centrosome duplication must occur in coordination with other cell cycle events, including DNA synthesis. The protease Separase, previously known to trigger sister chromatid separation, has been implicated in a licensing mechanism that restricts centrosome duplication to a single occurrence per cell cycle. Failure to properly control centrosome number results in supernumerary centrosomes, which are frequently found in cancer cells. Most human cancers exhibit centrosome duplication errors which might lead to aneuploidy and cancer formation. How centrioles are assembled and how their numbers are controlled within cells constitute long-standing unresolved questions.

Figure 3

Figure 3: Overexpression of Plk4 leads to the formation of multipolar mitotic spindles. A tetracycline-inducible Plk4 U2OS cell line was generated. Plk4 expression was induced through removal of tetracycline from the medium. Red: centrosomal staining (γ-tubulin), green microtubule staining (α-tubulin), blue (DAPI), Scale bar, 20 µm

The localization of a number of protein kinases including the polo-like kinases, Plks, to the centrosome has revealed the importance of protein phosphorylation in controlling the centrosome duplication cycle. These serine/threonine kinases were first described in mutants that failed to undergo a normal mitosis in Drosophila melanogaster (polo) and Saccharomyces cerevisiae. Subsequently, Plks have been found in many eukaryotes and have been shown to have key roles during entry into mitosis, bipolar spindle formation, chromosome segregation and cytokinesis. Mammalian cells express four polo-like kinase family members – Plk1, Plk2(Snk), Plk3(Fnk, Prk) and Plk4(Sak). All polo-like kinases consist of an N-terminal kinase domain and a conserved C-terminal region containing two polo-boxes except for Plk4 which contains only one polo-box. The polo-box domain is critical for Plk1 localization and function, since mutations in this region of Plk1 result in loss of Plk1 localization to the spindle poles, In particular, Plk2 and Plk4 kinases are involved in the regulation of centriole duplication (Warnke et al, 2004, Cizmecioglu et al., 2008 and Figure 2 and Figure 3).
Our aim is to identify and characterize substrates and regulators of Plk2 and Plk4 kinases in order to gain a better understanding on the mechanisms how centriole duplication is regulated. In addition, we are currently identifying novel centrosomal proteins by genome-wide siRNA screens in mammalian cells, yeast-two hybrid screens and biochemical approaches in normal and malignant cells.

To decipher signaling pathways involved in centrosome overduplication in cancer cells we use HPV (human papilloma virus)-induced cervial carcinogenesis as a model system. The high-risk human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Expression of the E7 oncoprotein rapidly drives centrosome duplication errors leading to aberrant centrosome numbers. The goal of our study is to decipher the cellular pathways and mechanisms of action of the high-risk HPV16-E7 oncoprotein leading to centrosomal abnormalities and subsequent genomic instability.

Figure 4: Regulation of the G2/M phase transition by Cdc25 phosphatases. In human cells there are three Cdc25 phosphatase family members, Cdc25A, B and C. All three are required for mitotic entry

Cdc25 phosphatases activate Cdk/Cyclin complexes by dephosphorylation and thus promote cell cycle progression. We recently observed that the peak activity of Cdc25A phosphatase precedes the one of Cdc25B coinciding with prophase and the maximum of Cyclin/Cdk kinase activity. Furthermore, Cdc25A activates both Cdk1-2/Cyclin A and Cdk1/Cyclin B complexes while Cdc25B seems to be involved only in activation of Cdk1/Cyclin B. Concomitantly, repression of Cdc25A led to a decrease in Cyclin A-associated kinase activity and attenuated Cdk1 activation. Our results indicate that Cdc25A acts before Cdc25B - at least in cancer cells, and has non-redundant functions in late G2/early M phase as a major regulator of Cyclin A/kinase complexes (Timofeev et al., 2009, Figure 4).
The Cdc25A protein is rapidly degraded in response to both UV-induced and -IR-induced DNA damage by the proteasome. In addition, the phosphatase activity of Cdc25A is inactivated following phosphorylation by the checkpoint kinases Chk1 and Chk2. This suggests that DNA damage could lead to an inactivation followed by degradation of Cdc25A in order to prevent the activation of Cyclin A/ Cdk2 or Cyclin E/Cdk2 complexes which both act as in vivo substrates of Cdc25A. Using two dimensional tryptic phosphopeptide mapping we were able to identify S75, S123 and S177 as phosphorylation sites targeted by Chk1 and Chk2 kinases in vitro. These phosphorylation sites were mutated to non-phosphorylatable alanine. We found that the Cdc25A S75A protein was not degraded after UV-irradiation. Taken together our data imply that Cdc25A is phosphorylated at different sites by Chk1 and Chk2 kinases and is dependent on the type of DNA damage (Hassepass et al., 2003, 2004).

Our research activities are currently supported by the Deutsche Krebshilfe, the BMFT and the Deutsche José Carreras Leukämie-Stiftung e.V.

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