Cyclin D

Introduction

Once the cells reach a critical cell size (and if no mating partner is present in yeast) and if growth factors and mitogens (for multicellular organism) or nutrients (for unicellular organism) are present, cells enter the cell cycle. In general, all stages of the cell cycle are chronologically separated in humans and are triggered by cyclin-Cdk complexes which are periodically expressed and partially redundant in function. Cyclins are eukaryotic proteins that form holoenzymes with cyclin-dependent protein kinases (Cdk), which they activate. The abundance of cyclins is generally regulated by protein synthesis and degradation through an APC/C dependent pathway.

Cyclin D is one of the major cyclins produced in terms of its functional importance. It interacts with four Cdks: Cdk2, 4, 5, and 6. In proliferating cells, cyclin D-Cdk4/6 complex accumulation is of great importance for cell cycle progression. Namely, cyclin D-Cdk4/6 complex partially phosphorylates retinoblastoma tumor suppressor protein (Rb), whose inhibition can induce expression of some genes (for example: cyclin E) important for S phase progression.

Mice, Drosophila and many other organisms only have one cyclin D protein. In humans, in addition to the mouse homologue, two more cyclin D proteins have been identified. These human proteins, called cyclin D1, cyclin D2, and cyclin D3 are expressed in most proliferating cells and the relative amounts expressed differ in various cell types.

Homologues

The most studied homologues of cyclin D are found in yeast and viruses.

The yeast homologue of cyclin D, referred to as Cln3, interacts with Cdc28 (cell division control protein) during G1.

In viruses, like Saimiriine herpesvirus 2 (Herpesvirus saimiri) and Human herpesvirus 8 (HHV-8/Kaposi's sarcoma-associated herpesvirus) cyclin D homologues have acquired new functions in order to manipulate the host cell’s metabolism to the viruses’ benefit. Viral cyclin D binds human Cdk6 and inhibits Rb by phosphorylating it, resulting in free transcription factors which result in protein transcription that promotes passage through G1 phase of the cell cycle. Other than Rb, viral cyclin D-Cdk6 complex also targets p27^Kip, a Cdk inhibitor of cyclin E and A. In addition, viral cyclin D-Cdk6 is resistant to Cdk inhibitors, such as p21^CIP1/WAF1 and p16^INK4a which in human cells inhibits Cdk4 by preventing it from forming an active complex with cyclin D.

Cyclins in humans

Growth factors stimulate the Ras/Raf/ERK that induce cyclin D production. One of the members of the pathways, MAPK activates a transcription factor Myc, which alters transcription of genes important in cell cycle, among which is cyclin D. In this way, cyclin D is synthesized as long as the growth factor is present.

Even though cyclin D levels in proliferating cells are sustained as long as the growth factors are present, a key player for G1/S transition is active cyclin D-Cdk4/6 complexes. Despite this, cyclin D has no effect on G1/S transition unless it forms a complex with Cdk 4 or 6.

One of the best known substrates of cyclin D/Cdk4 and -6 is the retinoblastoma tumor suppressor protein (Rb). Rb is an important regulator of genes responsible for progression through the cell cycle, in particular through G1/S phase.

In its un-phosphorylated form, Rb binds a member of E2F family of transcription factors which controls expression of several genes involved in cell cycle progression (example, cyclin E). Rb acts as a repressor, so in complex with E2F it prevents expression of E2F-regulated genes, and this inhibits cells from progressing through G1. Active cyclin D/Cdk4 and -6 inhibit Rb by partial phosphorylation, reducing its binding to E2F and thereby allowing E2F-mediated activation of the transcription of the cyclin E gene and the cell progresses towards S-phase. Subsequently, cyclin E/Cdk2 fully phosphorylates Rb and completes its inactivation.

Regulation in humans

Cyclin D is regulated by the downstream pathway of mitogen receptors via the Ras/MAP kinase and the β-catenin-Tcf/LEF pathways and PI3K. The MAP kinase ERK activates the downstream transcription factors Myc and AP-1 which in turn activate the transcription of the Cdk4, Cdk6 and cyclin D genes, and increase ribosome biogenesis. Rho family GTPases and focal adhesion kinase (FAK) activate cyclin D gene in response to integrin.

p27^kip1 and p21^cip1 are cyclin-dependent kinase inhibitors (CKIs) which negatively regulate CDKs. However they are also promoters of the cyclin D-CDK4/6 complex. Without p27 and p21, cyclin D levels are reduced and the complex is not formed at detectable levels.

In eukaryotes, overexpression of translation initiation factor 4E (eIF4E) leads to an increased level of cyclin D protein and increased amount of cyclin D mRNA outside of the nucleus. This is because eIF4E promotes the export of cyclin D mRNAs out of the nucleus.

Inhibition of cyclin D via i.a. inactivation or degradation leads to cell cycle exit and differentiation. Inactivation of cyclin D is triggered by several cyclin-dependent kinase inhibitor protein (CKIs) like the INK4 family (e.g. p14, p15, p16, p18). INK4 proteins are activated in response to hyperproliferative stress response that inhibits cell proliferation due to overexpression of e.g. Ras and Myc. Hence, INK4 binds to cyclin D- dependent CDKs and inactivates the whole complex. Glycogen synthase kinase three beta, GSK3β, causes Cyclin D degradation by inhibitory phosphorylation on threonine 286 of the Cyclin D protein. GSK3β is negatively controlled by the PI3K pathway in form of phosphorylation, which is one of several ways in which growth factors regulate cyclin D. Amount of cyclin D in the cell can also be regulated by transcriptional induction, stabilization of the protein, its translocation to the nucleus and its assembly with Cdk4 and Cdk6.

It has been shown that the inhibition of cyclin D (cyclin D1 and 2, in particular) could result from the induction of WAF1/CIP1/p21 protein by PDT. By inhibiting cyclin D, this induction also inhibits Ckd2 and 6. All these processes combined lead to an arrest of the cell in G0/G1 stage.

There are two ways in which DNA damage affects Cdks. Following DNA damage, cyclin D (cyclin D1) is rapidly and transiently degraded by the proteasome. This degradation causes release of p21 from Cdk4 complexes, which inactivates Cdk2 in a p53-independent manner. Another way in which DNA damage targets Cdks is p53-dependent induction of p21, which inhibits cyclin E-Cdk2 complex. In healthy cells, wild-type p53 is quickly degraded by the proteasome. However, DNA damage causes it to accumulate by making it more stable.

Regulation in yeast

A simplification in yeast is that all cyclins bind to the same Cdc subunit, the Cdc28. Cyclins in yeast are controlled by expression, inhibition via CKIs like Far1, and degradation by ubiquitin-mediated proteolysis.

Role in cancer

Given that many human cancers happen in response to errors in cell cycle regulation and in growth factor dependent intracellular pathways, involvement of cyclin D in cell cycle control and growth factor signaling makes it a possible oncogene. In normal cells overproduction of cyclin D shortens the duration of G1 phase only, and considering the importance of cyclin D in growth factor signaling, defects in its regulation could be responsible for absence of growth regulation in cancer cells. Uncontrolled production of cyclin D affects amounts of cyclin D-Cdk4 complex being formed, which can drive the cell through the G0/S checkpoint, even when the growth factors are not present.

Overexpression can happen in one of three ways: as a result of gene amplification, impaired protein degradation, or chromosomal translocation. Gene amplification is responsible for overproduction of cyclin D protein in bladder cancer and esophageal carcinoma, among others.

In cases of sarcomas, colorectal cancers and melanomas, cyclin D overproduction is noted, however, without the amplification of the chromosomal region that encodes it (chromosome 11q13, putative oncogene PRAD1, which has been identified as a translocation event in case of mantle cell lymphoma). In parathyroid adenoma, cyclin D hyper-production is caused by chromosomal translocation, which would place expression of cyclin D (more specifically, cyclin D1) under an inappropriate promoter, leading to overexpression. In this case, cyclin D gene has been translocated to the parathyroid hormone gene, and this event caused abnormal levels of cyclin D. The same mechanisms of overexpression of cyclin D is observed in some tumors of the antibody-producing B cells. Likewise, overexpression of cyclin D protein due to gene translocation is observed in human breast cancer.

Additionally, the development of cancer is also enhanced by the fact that retinoblastoma tumor suppressor protein (Rb), one of the key substrates of cyclin D-Cdk 4/6 complex, is quite frequently mutated in human tumors. In its active form, Rb prevents crossing of the G1 checkpoint by blocking transcription of genes responsible for advances in cell cycle. Cyclin D/Cdk4 complex phosphorylates Rb, which inactivates it and allows for the cell to go through the checkpoint. In the event of abnormal inactivation of Rb, in cancer cells, an important regulator of cell cycle progression is lost. When Rb is mutated, levels of cyclin D and p16INK4 are normal.

Another regulator of passage through G1 restriction point is Cdk inhibitor p16, which is encoded by INK4 gene. P16 functions in inactivating cyclin D/Cdk 4 complex. Thus, blocking transcription of INK4 gene would increase cyclin D/Cdk4 activity, which would in turn result in abnormal inactivation of Rb. On the other hand, in case of cyclin D in cancer cells (or loss of p16INK4) wild-type Rb is retained. Due to the importance of p16INK/cyclin D/Cdk4 or 6/Rb pathway in growth factor signaling, mutations in any of the players involved can give rise to cancer.

Mutant phenotype

Studies with mutants suggest that cyclins are positive regulators of cell cycle entry. In yeast, expression of any of the three G1 cyclins triggers cell cycle entry. Since cell cycle progression is related to cell size, mutations in Cyclin D and its homologues show a delay in cell cycle entry and thus, cells with variants in cyclin D have bigger than normal cell size at cell division.

p27⁻/⁻ knockout phenotype show an overproduction of cells because cyclin D is not inhibited anymore, while p27⁻/⁻ and cyclin D⁻/⁻ knockouts develop normally.