Cell cycle signaling network

DNA replication, DNA repair, cell division signaling and programmed cell death

The cell cycle is a highly regulated process and "takes micromanagement to the extreme". Various positive- and negative-feedback systems ensure that cells divide in a controlled manner. The process consists of a sequence of events by which a growing cell duplicates all its components and divides into two daughter cells, each with sufficient machinery to repeat the process. In eukaryotic cells, one round of cell division consists of two “gap” phases termed G₁- and G₂-, an S-phase during which duplication of all DNA happen, and an M-phase where proper segregation of duplicated chromosomes and chromatid separation occur. During each of these phases, regulatory signaling pathways monitor the successful completion of events in each phase before proceeding to the next phase. These regulatory pathways are commonly referred to as cell cycle checkpoints. Cell cycle checkpoints are activated in response the following (Figure 1):

• Cellular damage
• Exogenous cellular stress signals
• Lack of availability of nutrients, hormones and essential growth factors.

During the G₁ phase many signals intervene to influence cell division and the deployment of a cell’s developmental program (Figure 1). Crucial "decisions" are made to pass the G₁ restriction point as commitment to replicate DNA and divide is irreversible until the next G₁ phase. Failure to meet the correct conditions results in a failed attempt to divide. Signaling events converge to affect the phosphorylation status of the retinoblastoma protein (pRB) family (pRB, p107, and p130). Cyclin dependent kinases (CDKs) play a crucial role in pRB phosphorylation status and their activity is in turn controlled by cell stress and growth inhibitory signaling pathways. Sufficient phosphorylation (hyper-phosphorylation) of pRB causes it to dissociate from the elongation factor 2 (e2F) family of transcription factors. Dissociated e2F transcription factors mediate the transcription and activity of genes required for DNA replication during the S-phase.

As soon as the restriction point (G₁/S transition checkpoint) is passed, initiation of DNA replication takes place at multiple sites on the chromosomes, called the origins of replication. The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative complex (pre-RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10-dependent transcript (Cdt1), mini-chromosome maintenance (MCM) proteins, clamp-loaders, sliding clamps, helicases, DNA polymerases etc. The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once-per-cell-cycle and its binding to the chromatin marks the final step of pre-RC formation. Once the replisome is assembled, the transition to DNA replication is irreversibly completed and the cell enters the S-phase.

After successful completion of DNA replication the mitosis promoting factor (MPF) complex forms and plays a crucial role in nuclear envelope breakdown, centrosome separation, spindle assembly, chromosome condensation and Golgi fragmentation during mitosis. Cells only enter mitosis (G₂/M transition) after the completion of the above events.

When a cell is unable to address the above circumstances, cell division is permanently halted and the cell either enters senescence or programmed cell death is activated (Figure 1). Programmed cell death (particularly apoptosis) removes potentially hazardous cells from a population of cells, resulting in the controlled destruction of the cells designated for destruction. Two checkpoints during the cell cycle exist.

1. The DNA structure checkpoint
2. The spindle checkpoint

The DNA structure checkpoint operates between the G₁/S transition, the S-phase and the G₂/M transition (Figure 1). The DNA structure checkpoint during the G₁/S and G₂/M transitions ensure that DNA damage is minimal while the S-phase DNA structure checkpoint also recognizes and deals with replication intermediates, stalled replication forks and unreplicated DNA. Whenever the criteria are not met during a checkpoint, a cell will not proceed to the next phase. Various signaling networks are activated and operate to ensure these criteria are met. DNA structure checkpoint signaling has the same pattern during any phase of the cell cycle (Figure 1):

• Detection: Sensor proteins include proliferating cell nuclear antigen (PCNA)-like and replication factor C (RFC)-like protein complexes (see Sliding clamps, clamp-loaders and helicases), which are able to bind to damaged DNA to form a scaffold for downstream repair proteins. The Rad50/Mre11/NBS1 complex is also loaded onto damaged DNA sites and mediates downstream checkpoint and repair proteins.
• Signal transduction: Activated sensor proteins in turn activate several signaling proteins which in turn activates DNA repair mechanisms and downstream effector proteins that controls cell cycle checkpoint signal transduction and programmed cell death signaling. Some examples include, ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR) p53 binding protein (53bp), the topoisomerase binding protein TopBP1, mediator of DNA damage checkpoint (MDC1), breast cancer 1 (BRCA 1) etc.
• Effect: Downstream of the signal transducers include the the effector serine/threonine protein kinases CHK1 and CHK2. CHK’s transfer the signal of DNA damage to the phosphotyrosine phosphatases and cell division cycle proteins Cdc25A, Cdc25B, and Cdc25C as well the tumor-suppressor p53. Cdc25A controls the G₁/S and S-phase transition (prevents pRB dissociation through dephosphorylation of pRB proteins) while Cdc25B and Cdc25C control the G₂/M transition (both upregulating Wee1 and Myt1 by phosphorylation, which together control Cdc2/CyclinB activity). Tumor supressor p53 protein activity links DNA damage to programmed cell death.

Figure 1: Dynamic control of cell cycle events through cell signaling, checkpoints, nutrient availability and extracellular stress.

The spindle assembly checkpoint is a molecular system that ensures accurate segregation of mitotic chromosomes and functions during the M-phase of cell division. The spindle checkpoint depends on the activity of two systems.

1. The 26S proteasome (APC/C-cdc20 complex) for the degradation of cyclin B.
2. The anaphase promoting complex/cyclosome (APC/C-cdh1 complex) for the degradation of cyclins and securin

How are these for provocative sounding titles:
Voges D, Zwickl P, Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem. 1999;68:1015-68.
Peters JM. The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol. 2006 Sep;7(9):644-56.

Cyclin B is ubiquitinylated and degraded by the the 26S proteasome (APC/C-cdc20 complex) which in turn results in the activation of the APC/C-cdh1 complex. The APC/C-cdc20 complex is controlled by the mitotic checkpoint complex (MCC) which detects tubulin and kinetochore integrity. The APC/C-cdh1 complex mediates the degradation of securin resulting in chromosome segregation.

There is a considerable amount of cross-talk between DNA repair mechanisms, programmed cell cycle signaling pathways, cell death pathways (autophagy, apoptosis, mitotic catastrophe etc.) and other cell stress signaling pathways. All these intricately interwoven pathways serve to ensure accurate cell division and removal of faulty cells from a population through programmed cell death. The problem comes when one of the checkpoints or programmed cell death pathways become corrupted and causes uncontrolled cell division in multicellular organisms. Cancer is one of the outcomes of abrogated cell death signaling and uncontrolled cell division. Programmed cell death is however not limited to multicellular organisms as bacteria also contain the necessary pathways to self destruct.

E.g.:
Engelberg-Kulka H, Amitai S, Kolodkin-Gal I, Hazan R. Bacterial programmed cell death and multicellular behavior in bacteria. PLoS Genet. 2006 Oct;2(10):e135.

Rice KC, Bayles KW. Molecular control of bacterial death and lysis. Microbiol Mol Biol Rev. 2008 Mar;72(1):85-109.