Proteins regulating cyclin dependent kinases Cdk4 and Cdk5

The exact passage through the eukaryotic cell cycle is regulated by the progressive activation and inactivation of a family Cdk-s. Cancer cells evolve from normal cells when some essential processes in a dividing cell malfunction. This causes inappropriate replication, segregation and repair of the genome during progression of the cell cycle. Increased Cdk4 activity due to overexpression of Cdk4 and/or cyclin D, or because p16 INK4A, the Cdk4 specific inhibitor is missing from the cell, will cause the cell to cycle without a functional restriction point. The duration of the cell cycle shortens giving rise to mistakes in DNA replication. These malignant cells will grow out to form tumours.Potential antineoplastic drugs against certain forms of cancer could be synthetic chemicals which would inhibit for instance Cdk4 activity. Screening of compounds on their Cdk inhibitory effect is one example of how pharmaceutical industries are performing cancer research. In chapter 2 of the thesis a method is presented to facilitate Cdk4 compound screening. Human Cdk4 is expressed in the yeast Saccharomyces cerevisiae under the control of the GAL1-GAL10 promoter and inhibits cell growth when the yeast is grown on galactose. Coexpression of p16 INK4Arestores yeast cell growth. Moreover flavopiridol, the Cdk inhibitor which has already entered phase 3 clinical trials, is also restoring growth when it is added to the growth medium of the yeast. Simple OD readings of this yeast transformation when grown on galactose could give useful information upon whether a compound added to the growth medium could be a Cdk4 inhibitor or not. This Cdk4 inhibition will be of certain specificity since the yeast Cdc28 has high homology with human Cdk1 and Cdk2.Components, which normally would reside in the cell cycle, have recently been found to function atypically in non-proliferating neuronal cells. The cyclin dependent kinase Cdk5 for instance is identified via its homology with other Cdk-s and has not been shown to play a role in the cell cycle. Cdk5 has a ubiquitous tissue distribution in mammals with brains containing the highest amount of the transcript. On the other hand, the expression of p35, the only known activator of Cdk5, which has no homology to cyclins, is strictly confined to brains. Activated Cdk5 kinase phosphorylates a number of cytoskeletal proteins including neurofilaments and the neuron-specific microtubule associated protein tau in vitro , which are assumed to be the natural substrates. Phosphorylation of cytoskeletal proteins may play an important role in the polymerization and assembly of cytoskeletal elements which, in turn, may effect the growing neurites suggesting that Cdk5 is involved in the growth and maintenance of neurites. Recently Cdk5 has been shown to play a role in differentiation of muscles as well.Nerve cells are terminally differentiated before the onset of birth. Since nerves do not have any regeneration capacity, damaged or malignant cells will automatically undergo apoptosis. In the neurodegenerative diseases dementia (diffuse Lewy body disease), Parkinson's and amyotrophic lateral sclerosis neurofilament proteins are hyperphosphorylated whereas in Alzheimer's disease the tau protein is hyperphosphorylated. These malignant nerve cells undergo apoptosis leading to a certain death of the patient suffering from these diseases. Neurodegeneration possibly occurs as a result of inappropriate activation/deactivation of tissue-specific components of the cell cycle. It is very likely that Cdk5 is overactive in these malignant nerve cells.Since Cdk5 has been found in both proliferative and differentiated cells it is interesting to search for protein interactions with Cdk5 and the effect of these proteins on the Cdk5 kinase. During these investigations attempts were made to search for protein interactions with Cdk5 in proliferative cells and the possible roles of these proteins towards the Cdk5 kinase.In chapter 3 a C-terminal fragment of DNA binding protein, dbpA is described which can bind to Cdk5. This protein-protein interaction is shown via the yeast-two-hybrid system. Several in vitro experiments have confirmed that the C-terminal fragment of dbpA indeed specifically binds to Cdk5. Kinase assays have shown that this protein fragment inhibits the phosphorylation of both histone H1 and pRb by the Cdk5 kinase. DbpA is expressed in skeletal muscle and the heart. Since Cdk5 has been found to play a role in differentiation of muscles it is possible that dbpA plays a role in this phenomenon as well.Chapter 4 depicts another Cdk5 binding protein, a 60S ribosomal protein, L34. This protein interaction is discovered via the yeast-two-hybrid system as well. L34 like dbpA blocks the phosphorylation of histone H1 and pRb by Cdk5.Cloning of the cdk5 gene has been done via PCR on cDNA libraries. PCR on cDNA from a fetal brain library revealed two PCR products. One of the PCR products had the size of the cdk5 open reading frame whereas the other product was much smaller. Sequencing of both products showed the larger fragment coded indeed for the cdk5 gene whereas the other product was identical with the cdk5 open reading frame with bases 313 to 409 missing in the middle of the gene. In fetal brain this Cdk5 isoform could be expressed which lacks amino acids 105-136, which are thought to be responsible for catalyzing the phosphorylation reaction. Although this Cdk5 isoform (Cdk5i) can bind to p35, little kinase activity has been shown. Since this cdk5 variant has been found in total RNA of SH-SY-5Y neuroblastoma cells as well, but not in cDNA libraries of T-cells, HeLa cells, thymus, placenta and cerebellum, this protein could play a specific role in the differentiation of human nerve cells too. Binding of Cdk5i in these respective cells to p35 could prevent the wildtype Cdk5 from being activated. In this way Cdk5i could modulate the activity of the Cdk5 wildtype kinase. Chapter 5 depicts the results of this research.</p