AfCS Research Reports

Most diagrams in published papers are drawn using informal notations with sets of arrows, bar-headed lines, and circles roughly representing activation, inhibition, and the proteins involved, respectively. Fig. 2 is a typical example of just such a diagram for a MAPK cascade in a mammalian cell.

In this diagram, the arrows may implicate several different reactions. For example, the arrow from Ras to Raf (marked as 1 in Fig. 2) appears to indicate that Ras activates Raf. However, in reality, Ras enhances plasma membrane translocation of Raf. Thus, this arrow is more accurately read as recruitment or translocation, rather than activation. Two arrows originating from ERK to RSK and c-Myc (marked as 2 in Fig. 2) are interpreted as activation of RSK and c-Myc by ERK. However, the same representation could also be interpreted as one complex (ERK) that splits into two subcomponents (RSK and c-Myc). The reason that we exclude this interpretation is because we already know some of the properties of the components involved, not because of anything within the diagram itself. How should we interpret the arrow leading from RSK to RSK (marked as 3 in Fig. 2)? In this case, the arrow is meant to be read as the translocation of RSK from cytosol to nucleus, instead of activation of RSK by RSK itself. Therefore, among these simple examples, there are three possible interpretations of the same arrow symbol—activation, dissociation, and translocation.

Not only do notations in Fig. 2 have multiple meanings, they are ambiguous and unable to represent essential information (and therefore not machine readable). Correct interpretation depends upon the reader’s foreknowledge. For example, two arrows leading to Raf from PKC and Src indicate the activation of Raf by these two kinases. However, it is unclear what the mechanisms are, which residues are phosphorylated, or which is the first modulator of Raf. Accompanying text can supplement missing information to explain otherwise ambiguous points; however, in some cases the text can be more ambiguous than the diagrams.

Fig. 2. An example of an informal arrows-and-bars diagram. In this example, the same arrow pattern is used to represent three different reactions: activation, dissociation, or translocation, as follows. (1) The arrow (blue) from Ras to Raf indicates that Ras activates Raf. However, in reality, Ras enhances plasma membrane translocation of Raf. This arrow is more accurately read as “recruitment” or “translocation,” rather than activation. (2) Two arrows (light green) originating from ERK to RSK and c-Myc are interpreted as activation of RSK and c-Myc by ERK. However, the same representation could also be interpreted as one complex (ERK) that splits into two subcomponents (RSK and c-Myc). (3) The arrow (orange) is meant to indicate translocation of RSK from cytosol to nucleus rather than activation of RSK by RSK itself.

Kurt Kohn may have been the first to propose well-defined canonical representations for molecular interactions (2,3), and other researchers have been working on alternative representations (4-6). Unfortunately, none of the proposals has been widely used for a variety reasons. For example, there is no software tool to create a Kohn map efficiently, and this type of representation does not allow for explicit display of temporal processes. Other notations have different shortcomings.

Circuit schematics used in electronics are ideal examples of information display in a graphical but unambiguous manner. Engineers can reproduce the circuits drawn in the schematics simply from the information contained in the diagram. Although the interactions may be substantially more complex, one of our first goals in systems biology is to create standard graphical notations that unambiguously represent molecular interactions of biological systems.

A successful graphical notation system must (1) allow representation of diverse biological objects and interactions; (2) be semantically and visually unambiguous; (3) be able to incorporate notations; (4) allow tools to convert a graphically represented model into mathematical formulas for analysis and simulation; and (5) have software support to draw the diagrams. Although several graphical notation systems have already been proposed (2-6), each has obstacles to becoming a standard. Kitano proposed a graphical notation system for biological networks (7) designed to express sufficient information in a clearly visible and unambiguous way. Using these notations, the molecular interactions shown in Fig. 2 can be graphically represented as shown in Fig. 3.

Fig. 3. A process diagram with a consistently defined notation system. The notation system used by CellDesigner can describe biochemical reactions in more detail than that used in Fig. 2. The use of specific symbols helps to distinguish events such as activation, translocation, or dissociation. In addition, the specific biochemical state of a molecule can be defined.

The filled arrow (blue) in Fig. 2 is replaced by an open arrow and a circle-headed line in Fig. 3. The open arrow (blue) indicates translocation of Raf from cytosol to plasma membrane, and the circle-headed line (blue) from Ras to the open arrow indicates that Ras promotes translocation of Raf to plasma membrane, where Raf is fully activated via phosphorylation on both tyrosine-341 and serine-338 residues by Src and PKC, respectively. Indeed, the interaction of Ras with Raf is generally indicated by an arrow used for activation, but this process is actually the translocation of Raf, which is stimulated by Ras. Each of the two arrows (light green) originating from ERK to RSK and c-Myc in Fig. 2 is represented in a very different way in Fig. 3. The arrow heading to RSK is replaced by a circle-headed line that indicates that RSK is phosphorylated by ERK and subsequently stimulates its autophosphorylation. The three filled arrows (light green) between four RSK nodes indicate the state transitions caused by phosphorylation. Each state of phosphorylation can be described sequentially. On the other hand, the pathway from ERK to c-Myc is interpreted as ERK homodimer formation and translocation to the nucleus, where homodimerized ERK activates c-Myc. When the reaction is described in this manner, an interpretation such as “one complex (ERK) split into two subcomponents (RSK and c-Myc)” is impossible. The translocation of RSK from cytosol to nucleus is shown with the open arrow (orange) and can be easily distinguished from state transition or catalysis.

Overall, all reactions in Fig. 3 are easy to understand at a glance compared with the conventional informal notations. The notations also show specific characteristics of a protein. For example, readers can quickly recognize that SOS is a guanine nucleotide exchange factor for Ras just by looking at this diagram because sufficient information is presented. We believe that our notation system could be a convenient tool to enable researchers to share information involved in molecular interactions.

Fig. 3 is essentially a state transition diagram, often used in engineering and software development. This is an example of the process diagram, one of two representation modes, that allows the processes involved in the molecular interactions to be easily recognized. The alternative mode of visualization, the relationship diagram (not shown), is an extension of a Kohn map, where interactions for each molecular species are solidly represented but the temporal order of interactions is only implicit. Representation between the two display modes can be switched. Although these representations are unambiguous, they require software support (such as CellDesigner http://www.systems-biology.org/) to be drawn.

The symbols used to represent molecules and interactions are shown in Fig. 4 and Fig. 5. Each round-cornered box represents a specific state of a molecular species. The closed arrows (arrow head filled) represent changes in the state of modification (or allostericity) rather than indicating activation (as in Fig. 2). The schema avoids using symbols that directly point to the molecule to indicate activation and inhibition. Instead, the diagram directly indicates a transition from an inactive to an active state for activation and a transition from an active state to an inactive state for inhibition. When these transitions are promoted or inhibited by other mediating molecules, such as active kinases, these reactions are represented by circle-headed lines for activation and bar-headed lines for inhibition. An open arrow (arrow head not filled) indicates the translocation of a molecule.

Fig. 4. Main symbols adopted by CellDesigner version 2.0. These symbols are provided in CellDesigner version 2.0. Size and color of each module are changeable. CellDesigner also provides X-Y coordinates for each module and can distinguish between cellular compartments.

Fig. 5. Expression of the inner structures and states. The active state of the molecule is indicated by a dashed line surrounding the molecule. Although the example in Fig. 3 only describes phosphorylation as the possible alternative state, other state changes such as acetylation, ubiquitination, and allosteric changes can be represented with specific information such as target residue and position.