Sandbox Reserved 826

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.

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CDK4 in its biological contextCDK4 in its biological context

The cyclin-dependent kinase (CDK)4 forms the link between mitogenic and antimitogenic extracellular signals with the cell cycle and is located in the nucleus. As its name indicates, the assembly with a cyclin, in case of CDK4 a D-type cyclin, is prerequisite for the kinase activity of CDK4. Thus, in the active cyclin D-CDK4 complex, the D-type cyclin acts as a regulatory subunit that directs its complex partner to the retinoblastoma protein (pRB) substrate. pRB, the only physiologically important target of the cyclin D-CDK4 complex, is implicated in the coupling of the cell cycle clock with the transcription machinery. Thereby, it controls the passing of the restriction point in the G1 phase of the cell cycle. This G1 restriction point is the moment at which the cell commits whether it will proceed the cell cycle and proliferate by going towards the S phase or if potential DNA damage and metabolic disturbance have to be solved at first. In its hypophosphorylated state, pRB binds to the transcription factor E2F and inhibits it from initiating transcription of a number of genes that are important for cell growth control . In contrast, phosphorylation of pRB by CDK4 prevents pRB from binding to E2F, thereby allowing E2F to proceed with the activation of its regulated genes . Thus, it can be resumed that kinase activity of cyclin D-CDK4 allows transcription initiation of cell growth genes by phosphorylation of the E2F-inhibitor pRB .

Regulation of CDK4Regulation of CDK4

The activation of CDK4 includes several different and independent regulatory steps allowing CDK4 to integrate the various signals implicated in cell growth and proliferation. This comprises the assembly of cyclin D-CDK4 complexes, the nuclear translocation of D-type cyclin-CDK4 complexes, activity regulation, and phosphorylation of CDK4. At first, the most obvious regulation concerns the presence of both partners, CDK4 and the D-type cyclin, to be able to form a functional complex at all. This step also shows the inducibility of CDK4 by external growth signals. Extracellular mitogens induce the expression of the D-type cyclins required for cyclin D-CDK4 complex formation and thus, prevention of transcription factor inhibition by pRB. The concentration of the D-type cyclins has to pass an inhibitory threshold set by INK4 CDK4 inhibitory proteins that bind the isolated CDK4 thereby preventing the binding of cyclin. This already presents a possibility of negative regulation as growth inhibition signals may lead to an increasing accumulation of these inhibitory proteins. So, at this point, the key position of CDK4 for linking external signals with expression changes leading to cell cycle progress becomes clear. After a functional complex is formed, activity of cyclin D-CDK4 can further be regulated on several levels. On one hand, this can take place by altering the stability of the complex by supplementary binding components. One example might be the Cip/Kip CDK inhibitors (p21, p27) which paradoxically have been shown to stabilize the complexes cyclin D3-CDK4 and cyclin D1-CDK4 . Nevertheless, they are not essentially required for the formation of these complexes. Furthermore, as the pRB substrate of cyclin D-CDK4 and the CDK-activating kinase (CAK) are nuclear proteins, the complex has to be imported into the nucleus. This is mediated by the CDK4 binding proteins p21 and p27, which, in contrast to the complex partners, possess an obvious nuclear localization signal. Therefore, p21 and p27 are required to determine this translocation of the cyclin D-CDK4 complex, thereby constituting another regulation possibility. The Cip/Kip CDK inhibitors p21 and p27 can, as their names imply, inhibit the activity of the D-type cyclin-CDK4 complex. The underlying mechanisms are not understood yet, but it is supposed that the opposite effects of p21 and p27 on the CDK complex might depend on the relative stoichiometric ratio of CDK inhibitor and D-type cyclin . It can be retained that this inhibition represents another level of CDK4 activity regulation. To be catalytically active, CDK4 of the complex has to be phosphorylated on a specific residue within its activation loop. This phosphorylation only takes place if CDK4 is present in form of the complex with cyclin D. The catalysing enzyme is the CDK-activating kinase (CAK) which is interestingly the cyclin H-CDK7 complex. However, as CDK7 has been found to be constitutively active , it does not seem as CAK would regulate CDK4 specifically in response to mitogenic stimulations. According to some observations, an increased binding of p27 to the cyclin D-CDK4 complex may prevent phosphorylation, but these results are in contradiction with more recent observations that exclude the prevention of the activating phosphorylation of CDK4 as activity inhibition of cyclin D-CDK4 by p27 . To conclude, the activity of CDK4 can be regulated at several steps that are independently from each other. It is this complex process of regulation by integrating multiple signals and breaking them down on one point, the activity of CDK4 that allows the cell to respond in a simple manner – proliferation or not - to a variation of stimuli.

CDK4’s role in cancerCDK4’s role in cancer

A main characteristic of cancerous cells is the dysregulation of the cell cycle allowing uncontrolled cell growth and proliferation while safety mechanisms as senescence and apoptosis are inhibited. As the activation of the CDK4 pathway leads to transition from the G1 to the S phase of the cell-cycle via the inhibition of the retinoblastoma protein, this pathway plays an important role in cancerogenesis. In accordance with this idea, the CDK4 pathway was found to be altered in regulation in about 90% of studied melanomas . In case of cancer variants where the dysregulation of the CDK4 signalling pathway is the main reason for melanoma development and not a secondary effect, drugs that target and inhibit CDK4 might be very promising. Therefore, further studies should be undertaken to develop new therapeutic agents fighting cancer at this central point of cell-cycle regulation.