Collaboration of Mitotic Kinases in Cell Cycle Control

The cell cycle allows faithful inheritance of the genetic material from mother to daughter cells. Cell cycle events are controlled tightly by several kinase families including CDKs, Plks, and Aurora kinases. What is interesting about these kinases is that they don't always act alone. They sometimes collaborate with each other, making the regulatory network much more complex but also more accurate.

Why is protein phosphorylation important? Phosphorylation is a universal protein modification in eukaryotic cells, and participates in every aspect of cellular life. The human genome encodes over 500 protein kinases that interact with diverse substrates including other kinases, transcription factors, and regulatory proteins. The cell cycle is no exception, and is tightly regulated by protein phosphorylation. Substrates are phosphorylated in a spatiotemporally specific manner by protein kinases on serine, threonine, or tyrosine residues that can subsequently be recognized by other proteins that contain a "phosphopeptide binding domain." In this way, signaling cascades initiated by protein phosphorylation trigger a series of changes in protein-protein interactions. The assembly or disassembly of protein complexes takes place at a specific time and location within the cell, leading to turning a signaling pathway "on" or "off" (Figure 1).

Decades ago, scientists discovered cyclin-dependent kinases (CDKs), proteins that act as master regulators of the cell cycle. As their name indicates, the activity of CDKs is controlled by cyclins, other proteins whose expression level oscillates throughout the cell cycle. Different CDKs operate during different phases of the cell cycle and provide the driving force for cell cycle progression. Besides CDKs, scientists have found several protein kinases that also control the cell cycle, primarily Plks (Polo-Like kinases) and Aurora kinases. These kinases control protein phosphorylation in a spatiotemporal manner — meaning they control where and when phosphorylation happens. In fact, these kinases are major regulators of centrosome function, spindle assembly, chromosome segregation, and cytokinesis. Unlike CDKs, which promote cell cycle progression, these kinases are responsible for avoiding errors during cell cycle progression (Figure 2; Archambault & Glover 2009, Hochegger et al. 2008, Fu et al. 2007, Barr et al. 2004, Carmena et al. 2003).

How do scientists study protein phosphorylation? They use powerful tools developed by structural biologists to understand the mechanics of protein phosphorylation. The interaction between kinase and substrate is quite dynamic, and therefore difficult to follow. But under proper conditions, protein kinases form complexes with peptides derived from substrates. In order to maintain a stable kinase-substrate complex for analysis, scientists use ATPγS (a non-hydrolyzable substrate for ATPases) in a cellular system. This blocks the phosphoryl transfer and the phosphorylation process. With this blockage, the resulting crystal structures of the kinase together with its substrate in active conformations are quite informative. Their structure and binding shapes can be then analyzed by biologists.

Also, we now know that the active conformations of many protein kinases are similar, and so we can classify groups of kinases based on their similar structure. For example, CDK2 and Aurora kinase possess a two-lobe architecture in their kinase domain: the N-terminal lobe and the C-terminal lobe. The active site of these kinases is buried at the interface of these two lobes, which forms a kind of groove, and also includes the ATP binding site and the kinase activation segment (Figure 3; Jeffrey et al. 1995; Fu et al. 2007).

How does the kinase-substrate interaction facilitate phosphorylation? First, the kinase has to properly position the catalytic site and ATP, and then correctly orient the substrate binding site. The kinase itself needs to be phosphorylated at a threonine residue within the activation segment. The docking portion of the substrate then fits into the groove on the surface. Then the kinase transfers a phosphate group from the ATP onto the Ser/Thr/Tyr of the substrate. Protein kinases usually get activated by binding to other entities, called activators. For example, in the absence of the activator TPX2, the activation segment of Aurora A overlaps with the substrate binding site, so that substrate binding is not optimal. With the binding of TPX2, the two lobes of Aurora A kinase domain switch to a conformation that is suitable for substrate binding and phosphoryl transfer. (Fu et al. 2007). Similarly, CDK2 also requires an interaction with Cyclin A to be fully activated (Jeffrey et al. 1995).

Although scientists have known for some time that CDKs, Plks, and Aurora kinases play central roles in cell cycle regulation, they only recently began to consider possible crosstalk among their three signaling pathways.

Protein phosphorylation is a common way to regulate signaling pathways in the cell cycle. Kinases catalyze phosphoryl transfer from ATP to substrates and change downstream protein-protein interaction in such way that a signaling pathway is either switched on or shut off. Scientists have established the central roles of CDKs, Plks, and Aurora kinases in cell cycle regulation. Whereas CDKs provide the driving force for cell cycle progression, Plks and Aurora kinases function in a spatiotemporal manner to ensure correct cell division. These major mitotic kinases have attracted great attention in the cell cycle field, and their signaling pathways are each active subjects of research. Importantly, the emerging evidence of collaboration among these proteins uncovers a novel view of their complex regulation network both in time and space, and advances our understanding of cell cycle control.