Introduction The cell cycle is regulated by the activities of cyclin-dependent kinases (CDKs). In mammals, 11 different CDKs have been identified whereas in yeast only one major CDK drives the cell cycle. Of the 11 mammalian CDKs, CDK4 and CDK6 promote entry and progression through G1, CDK2 functions in entry and progression through S-phase, CDC2 (CDK1) regulates M-phase, and the other CDKs function in transcription or have unknown functions. The activities of CDKs are regulated by protein-protein interactions (with cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, subcellular localization, and multiple phosphorylations. Several functions for CDK2 have been suggested, including entry into S-phase by Rb phosphorylation, initiation of DNA replication, exit from S-phase, and progression through G2 phase. We are investigating the functions of CDK2 in an in vivo model system as well as in vitro by screening for inhibitors that prevent CDK2 activation.
Aims - Aim 1: Determination of the functions of CDK2 in a mouse model system. - Aim 2: Identification of inhibitors of CDK2 activation. - Aim 3: Search for CDK6 binding proteins in different tissues of the mouse. - Aim 4: Proteonomic approach to identify down stream targets in knock-out mice.
Aim 1 : Determination of the functions of CDK2 in a mouse model system. We are generating a conditional CDK2 knock-out mouse to study the functions of CDK2 in different tissues or at different times in the mouse development. In order to create such mice, we are isolating bacteria artificial chromosome (BAC) clones containing mouse CDK2. Currently, we have isolated four independent BAC clones containing CDK2. Next we have to sequence the CDK2 locus including at least 5 kb flanking region. Knowing the sequence of CDK2, we will create the targeting vectors for our knock-out mice. We will introduce the required elements (loxP sites, selectable maker [NEO cassette]) directly into a BAC clone, from which the final targeting vectors are derived. The knock-out facility at NCI will use these vectors to create mutant mice for us. Once we receive our mutant mice, we will need to breed them to homozygocity before the gene knock-out is induced by expressing the Cre-recombinase in the tissue(s) desired. We will then analyze the phenotype of CDK2-/- mice starting with the embryonic development and later expanding to adult mouse tissues. As a complimentary approach we have created transgenic mice that overexpress a dominant-negative mutant of CDK2 (D145N) from the human b-actin promoter. CDK2D145N when expressed in mammalian cell lines leads to G1 arrest with low CDK2 activity. We hope to down-regulate or inhibit the activity of CDK2 in the mouse or at least in certain tissues. Such transgenic mice have been just generated in our laboratory and we plan to breed them to homozygocity. Once we have homozygotes we will analyze these mice for their CDK2 activity in embryonic development and, if these transgenics are viable in adult tissues.
Aim 2 : Identification of inhibitors of CDK2 activation. Activation of CDK2 requires minimally two steps: cyclin binding and phosphorylation of threonine160 by the CDK-activating kinase (CAK). Therefore if we can prevent phosphorylation of CDK2 at Thr160, CDK2 will remain inactive. In order to screen for compounds that prevent CDK2 phosphorylation, we need an appropriate assay. We have generated highly specific antibodies that recognize CDK2 only when it is phosphorylated by CAK. Adding CAK to CDK2 in the presence of ATP creates an epitope that is readily detected by our phosphospecific antibody. In the presence of a candidate inhibitor, the antibody will not be able to detect CDK2 since it will remain unphosphorylated. This is the basis for a screen in 96-well plates to identify inhibitors from a library of natural products (in collaboration with the Drug Discovery Group at NCI). Once we have candidates, we will screen them in cell lines and possibly in animal models to investigate their potency and specificity. Inhibitors of CDK2 activation will not only be useful for basic research but could also be tested for tumors with elevated CDK2 activity.
Aim 3 : Search for CDK6 binding proteins in different tissues of the mouse. We are generating transgenic mice that express CDK6 with C-terminal His6 and Myc9 tags. These two tags will allow us to purify CDK6 from different mouse tissues with sufficient purity for masspectrometry analysis. Such analysis will identify proteins that are bound to CDK6 in a certain tissue or under certain conditions. Comparing proteins bound to CDK6 in different tissues will help us to determine if CDK6 is differentially regulated in different tissues. It is well know that some CDKs are differentially expressed in different tissues but we have no knowledge about differential regulation of these CDKs.
Aim 4 : Proteonomic approach to identify down stream targets in knock-out mice. When we generate knock-out (or transgenic) mice, we often observe a phenotype. Nevertheless, the pathways leading to a phenotype are complicated and we lack the knowledge of the down-stream targets of our mutated gene. Therefore, it would be very valuable to learn more about changes of protein expression in mutant mice. Our approach is to analyze whole mouse embryos on two-dimensional isoelectric focusing gels and compare wild-type embryos to mutant embryos. If we are able to detect spots that are differentially expressed in the two embryos, we are able to identify these proteins directly by masspectrometry. Once we have identified such proteins, we can study their mRNA expression and by using antibodies their expression and localization in different tissues. This is a complimentary approach to profiling of mRNA expression by using microarrays.
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