G1-Phase

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Now that we know more about the regulation of CDKs we can go into the details of the G1-phase regulation. Normal growing cells can enter the G1-phase either from the G0-phase by stimulating agents such as growth factors or from M-phase after one cell cycle. Terminally differentiated cells and quiescent cells are in the G0-phase but terminally differentiated cells are unable to enter the G1-phase unless they become cancer cells. The main regulatory proteins of the cell cycle are the CDKs as we learnt in the previous chapter (Cyclin-dependent Kinases). To refresh your memory, they are protein kinases and consist of a catalytic subunit and a regulatory subunit, a cyclin. The cyclin, which is present in early G1-phase is cyclin D1 (main D cyclin is D1, depending on cell type), which is also synthesized when cells enter the G1-phase coming from the G0-phase. When normal cells ´move´ through the G1-phase, they require the presence of growth factors in their environment and they require a certain rate of protein synthesis. Once cells pass through a point called the Restriction point they are determined to enter the S-phase and do not require growth factors any more until after one cell cycle. The restriction point, which is one of the checkpoints in the cell cycle, is inactivated more or less in cancer cells and later we will see how the inactivation occurs.

Let´s see how this looks like in an animation. Play around with the buttons and try to cause cell cycle arrest before and after the restriction point.

Let´s move on now. Cyclin D1 and the CDKs CDK4 and CDK6 form a complex and for the complex formation p27kip1 is required (see previous chapter). CDK4/6 like other CDKs are protein kinases adding a phosphate group to a protein. The only one protein, which is known to be phosphorylated by CDK4/6 is the Retinoblastoma protein or pRb. This protein is a tumor suppressor protein and has been found to be inactivated in basically all tumor cells by different types of mechanisms as we will see later. A tumor suppressor gene normally codes for a checkpoint protein and therefore, to inactivate these proteins mutations in both gene copies have to occur (recessive). Oncogenes such as RAS for example unlike tumor suppressor genes require only one mutation in one gene copy for tumor formation, because the mutant proteins are dominant (see chapter mutation). The pRb protein was originally found in a childhood cancer called Retinoblastoma, which is an inherited cancer of the eye. It was found that both gene copies are inactivated in children with retinoblastoma, which is typical for a tumor suppressor gene.

The retinoblastoma protein can be phosphorylated at several sites though uniquely at one site by CDK4/6 and at other sites by CDK2. In its unphosphorylated status the pRb is active and has domains, at which proteins can bind, which prevent transcription of genes for cell cycle progression. One protein is the transcription factor E2F, which is required for the gene expression of many important S-phase proteins. At a different site of pRb acomplex of proteins consisting of RBP1, a linker protein, to which enzymes called Histone deacetylases (HDAC) bind. These enzymes prevent the opening of the DNA surrounding protein complex and thus preventing transcription. You see how pRb works? It binds transcription factors such as E2F, which locate pRb together with the RBP1-HDAC protein complex and transcription is inhibited (Check out transcription again if you need to refresh your memory).

CDK4/6 now adds one phosphate group onto the pRb protein. This causes the release of of E2F and the RPBP1-HDAC complex and the synthesis of another cyclin, cyclin E, occurs. Cyclin E then forms a complex with CDK2. CDK2 also phosphorylates the pRb but at different sites of the protein. This leads to the complete inactivation of pRb. E2F now activates the transcription of S-phase required genes and CDK2 phosphorylates a number of different substrates, which is required for S-phase progression. Let´s have a look at a little movie. Start the movie by pushing the Start button.

In order to enter S-phase, several proteins are required to be phosphorylated by CDK2 such as a transcription factor specific for histones. Histones are proteins which cover the DNA and are only synthesized newly in S-phase. CDK2 also phosphorylates its own inhibitor, p27. You wonder how that can be? There is probably always some small amount of active CDK2 present, when the cyclin E level is high and when the p27 level is not further increased to inhibit CDK2. This is all a matter of balance. Cells which are entering S-phase synthesize a new cyclin, cyclin A which can also form a complex with CDK2 replacing cyclin E. Cyclin A can also form a complex with another CDK, CDK1 (formerly CDC2). Cyclin E at this point is destroyed signalling the end of G1-phase. In conclusion there are two checkpoints in G1-phase: the CDK4/6 and the CDK2 checkpoint. It is not surprising that cancer cells try to overcome these checkpoints to grow unregulated. We will hear more about this in the next chapter.

What comes next? In the next chapter we will see how this pathway is inhibited in normal cells and what are the mechanisms of inactivation in cancer cells.