Rb1 And The Cell Cycle

The product of the RB1 gene, ppHO™1, is most of all a central regulator of the cell cycle (Figure 5.3). The RB1 protein controls the transition from the G1 to the S phase by binding to E2F1, E2F2, or E2F3 proteins and thereby repressing the promoters of genes needed for the entrance into S phase. This repression is relieved and binding to E2F alleviated when ppHO™1 becomes hyperphosphorylated5 towards the end of G1. At least two successive phosphorylations are needed to inactivate RB1. Normally, the first phosphorylation is performed by a CDK4/Cyclin D holoenzyme, and is followed by further phosphorylations by the CDK2/Cyclin E holoenzyme. Hyperphosphorylated RB1 is inactive as far as G1/S cell cycle regulation is concerned. The protein likely has also functions in the S phase, where it may be involved in chromatin organization, and during mitosis, where it may help to organize proteins for chromosome segregation. Following mitosis, RB1 is partly dephosphorylated and its hypophosphorylated state restored. In this fashion, RB1 switches between hypophosphorylated and hyperphosphorylated states during the cell cycle.

5 The RB1 protein is always phosphorylated at some sites; therefore its phosphorylation varies between hypo- and hyperphosphorylation rather than between non-phosphorylated and phosphorylated. Up to 14 phosphates can be incorporated; their individual functions are not fully elucidated (understandably...).

Clearly, therefore, loss of RB1 function, at a minimum, upsets cell cycle regulation and may lead to unrestrained cell proliferation. Specifically, in the absence of RB1 immature cells such as retinoblasts may not spend sufficient time in G1 to enter a differentiated state or establish stable quiescence, i.e. G0. Worse, since RB1 may also be required for proper chromatin structure and chromosome segregation, cells may tend to become genomically instable and acquire additional alterations that favor tumor progression. At least one mechanism may protect against loss of RB1 function: Over-activity of E2F factors, particularly of E2F1, can induce apoptosis.

The mechanism of cell cycle regulation sketched so far is in fact much more complex and consists of several layers of control, even when only the G1 to S transition is considered (Figure 5.4). The activity of the CDK4 and CDK2 protein kinases depends strictly on the presence of their regulatory subunits, i.e., D-Cyclins and Cyclin E, respectively. Both fluctuate in a coordinate fashion in the course of the cell cycle. Cyclin D expression is directly dependent on stimulation by exogenous signals such as growth factors (^6.4). Moreover, CDKs are only active, if they are phosphorylated in a certain pattern. Phosphorylation at one specific threonine residue by the CDK activating kinase (CAK, identical to CDK7) in conjunction with its regulatory subunit Cyclin H is activating, while phosphorylation at two different threonines is inhibitory. The phosphates at these sites are removed by CDC25 phosphatases, which also respond to external signals (^6.4).

A further layer of control is provided by protein inhibitors of CDKs (Table 5.2). There are two classes of such inhibitors, the CIP/KIP and the INK proteins. The first comprises the p21CIP1, p27KIP1, and p57KIP2 proteins, the second p15INK4B, p16INK4A, p18INK4C, and p19INK4D. Their genes are now systematically designated CDKN1A -CDKN1C and CDKN2A - CDKN2D. All proteins are named according to their molecular weights. The function of the INK4 (inhibitor of kinase 4) proteins is straightforward. They compete with D-Cyclins for binding to CDK4 and block its kinase activity.

Figure 5.3 Function of RB1 in cell cycle regulation DP1 is a heterodimer partner of E2F proteins. HDAC: histone deacetylase; HAT: histone acetyl transferase. The functions of HDACs and HATs are explained in 8.4.

The function of the CIP/KIP proteins (CDK/kinase inhibitory proteins) is more complicated. At high concentrations, they inhibit the activity of CDKs in general, but different from INK4s they block the CDK-Cyclin holoenzymes. At moderate concentrations, they rather stimulate the assembly of CDK-Cyclin complexes. In proliferating cells, CIP/KIP proteins, in particular p27KIP1, help to coordinate the cell cycle. Until late G1, p27KIP1 is bound to the CDK2/Cyclin E complex delaying its activity until late G1 when CDK2 activity is required to inactivate RB1. At this point, the p27KIP1 inhibitor is phosphorylated and rapidly degraded (cf. 6.4). On the other hand, in cells that are not supposed to proliferate, high levels of inhibitor proteins arrest the cell cycle.

The different CDK inhibitors respond to different signals that lead to cell arrest, allowing fine-tuned cellular responses to different signals. For instance, in some cell

Figure 5.4 Three layers of cell cycle regulation The figure focuses on the G1^S transition, which is the usual point of regulation in human cells (mechanisms regulating G2^S transition are used in specific cells). The inner layer consists of the RB1 phosphorylation cycle which determines E2F activity. It is dependent on the second layer formed by the CDK/cyclin cycle (CDC2 is also named CDK1). This layer is additionally regulated by a third one involving phosphorylation and dephosphorylation of the CDKs (only one example is shown) and CDK inhibitor proteins. This layer interacts mutually with the inner layers, and is influenced by various mitogenic and anti-proliferative signals.

Figure 5.4 Three layers of cell cycle regulation The figure focuses on the G1^S transition, which is the usual point of regulation in human cells (mechanisms regulating G2^S transition are used in specific cells). The inner layer consists of the RB1 phosphorylation cycle which determines E2F activity. It is dependent on the second layer formed by the CDK/cyclin cycle (CDC2 is also named CDK1). This layer is additionally regulated by a third one involving phosphorylation and dephosphorylation of the CDKs (only one example is shown) and CDK inhibitor proteins. This layer interacts mutually with the inner layers, and is influenced by various mitogenic and anti-proliferative signals.

types p15INK4B is induced by inhibitory growth factors like TGFp. High levels of the inhibitor protein dissociate Cyclin D/CDK4 complexes and block CDK4. They also redirect any p21CIP1 or p27KIP1 present towards CDK2/Cyclin E, blocking this kinase as well. By comparison, p 16INK4A has a very slow turnover and accumulates gradually in continuously proliferating cells until its concentration becomes high enough to slow down the cell cycle or arrest it irreversibly (^7.4). p21CIP1 accumulates in a similar fashion during frequent replication, but also in response to various growth factors and to DNA damage (^5.3). p57KIP2 is expressed in a restricted range of cell types, most prominently during embryonic development where the protein appears to help establish a terminally differentiated state in specific tissues. The gene encoding p57KIP2, CDKN1C, is as a rule only expressed from the maternally inherited allele, i.e. it is imprinted (^8.2).

In summary, therefore, RB1 forms a node in the cell cycle regulation network which ensures that cell proliferation occurs only in response to proper sets of signals, e.g. following stimulation by growth factors via the MAPK and further signaling pathways (^6.4). It is easy to imagine how this regulation network becomes disrupted by loss of function of the central RB1 protein. Alternatively to loss of RB1 function itself, other components of the regulatory network may be affected in human cancers. So, overexpression of D-Cyclins or CDK4 as a consequence of gene amplification, or mutations of CDK4 that make it unresponsive to CDK inhibitors may exert similar effects.

In addition, alterations in CDK inhibitors are found in a variety of human cancers. In a wide range of human cancers, the CDKN2A gene is inactivated by point mutation, promoter hypermethylation, or homozygous deletion. In fact, CDKN2A must also be regarded as a 'classical' tumor suppressor gene, since both alleles are affected in such cancers, and germ-line mutations of the gene have been found in families prone to pancreatic cancer and melanoma (^12.4). Of note, not all these changes may be equivalent. There is some evidence that loss of RB1 function may be the most severe defect, as one might guess intuitively (^14.2).

Table 5.2. Inhibitor proteins of cyclin-dependent kinases

CDK Inhibitor

Gene

Location

CDK inhibited

Inducers

p21CIP1

0 0

Post a comment