Regulation of Cyclin Dependent Kinase 4 (CDK4)

Unrestricted entry into the cell cycle and abrogation of DNA damage checkpoints are both hallmarks of tumour progression. Tyrosine phosphorylation at position 17 of CDK4 appears to play a role in these phenomena, but so far the regulatory mechanisms behind this event remain uncharacterised. We are therefore interested in identifying the key players in this process, as this would enhance our understanding of the role and regulation of tyrosine phosphorylation on CDK4 in cell proliferation and DNA damage checkpoints. In addition, this approach may uncover new anticancer drug targets. The strategy taken has been two-fold: (i) to isolate and identify kinase(s) present in human cell lysates that are capable of phosphorylating Y17 of CDK4 and (ii) to investigate a role for Y17 phosphorylation in cell cycle control. Currently our studies have led to the identification of a novel kinase activity that can phosphorylate CDK4 on tyrosine 17. Using column chromatography techniques we have purified and obtained protein sequence of a candidate Y17 CDK4 kinase. This kinase appears to be distinct from the Wee1 kinase, which phosphorylates the analogous site (tyrosine 15) on CDK1. We are currently evaluating this candidate both biochemically and in the cell, as a Y17 CDK4 kinase.

Studies on Cyclin D1 Turnover

The product of the PRAD1 oncogene, cyclin D1 is a key regulator of the G1 phase of the cell division cycle where it binds to and activates the cyclin-dependent kinases CDK4 and CDK6. The activity of these complexes is under exquisite control, major contributions to this regulation being the subcellular localisation of cyclin D1 protein and its ubiquitin-mediated proteolysis via the 26S proteasome. Phosphorylation of threonine 286 (T286) on cyclin D1 is thought to regulate both localisation and turnover of the protein under certain cellular conditions.

Using biochemical fractionation by column chromatography, we have identified a kinase that can phosphorylate the T286 of cyclin D1 and may be a regulator of cyclin D1 turnover during the normal cell division cycle. Further evaluation of this kinase in the context of T286 cyclin D1 phosphorylation and turnover is ongoing. We have also investigated regulation of cyclin D1 turnover in the context of DNA damage. From theses studies we have discovered that T286 phosphorylation of cyclin D1 is induced in response to DNA damage and requires the activity of the CHK1 cell cycle checkpoint kinase. This would suggest a role for a CHK1-mediated DNA damage response in regulation of cyclin D1 turnover.