Representation of intracellular signaling networks as directed graphs allows for the identification of regulatory motifs. Regulatory motifs are groups of nodes with the same connectivity structure, capable of processing information. The bifan motif, made of two source nodes directly cross-regulating two target nodes, is an over-represented motif in a mammalian cell signaling network and in transcriptional networks. One example of a bifan is the two MAP-kinases, p38 and JNK that phosphorylate and activate the two transcription factors ATF2 and Elk-1. We have used a system of coupled ordinary differential equations to analyze the regulatory capability of this bifan motif by itself, and when it interacts with other motifs such as positive and negative feedback loops. Our results indicate that bifans provide temporal regulation of signal propagation and act as signal sorters, filters, and synchronizers. Bifans that have OR gate configurations show rapid responses while AND gate bifans can introduce delays and allow prolongation of signal outputs. Bifans that are AND gates can filter noisy signal inputs. The p38/JNK-ATF2/Elk-1bifan synchronizes the output of activated transcription factors. Synchronization is a robust property of bifans and is exhibited even when the bifan is adjacent to a positive feedback loop. The presence of the bifan promotes the transcription and translation of the dual specificity protein phosphatase MKP-1 that inhibits p38 and JNK thus enabling a negative feedback loop. These results indicate that bifan motifs in cell signaling networks can contribute to signal processing capability both intrinsically and by enabling the functions of other regulatory motifs.
Systematic understanding of the design principles of regulatory circuits in cells is a necessary step towards building predictive models of mammalian cells (1,2). Complex large-scale biochemical networks can be represented as graphs where nodes are molecular components and links represent their interactions (3,4). Analysis of such networks allows for the identification of motifs, reoccurring sub-graphs of few interacting nodes (5)(6)(7). In cell signaling networks and gene regulatory networks, regulatory motifs such as feed-forward (5,(8)(9)(10), feedback loops (11,12), single-input modules (SIM) (13,14) and bifans (9,11,15), represent small circuits with information processing capabilities that can alter input/output relationships within the network. Quantitative analysis of these motifs can be used to identify the information processing capabilities of these circuits. Several studies characterizing the dynamical behavior of network motifs such as feed-forward (8,16,17), feedback loops (18)(19)(20)(21), and bifans (22), as well as stability analysis of different motifs (23) have been published. These studies reported that feed forward motifs with AND configuration can exhibit a delay following by signal activation (8,13,16,24) and filter noise (17,25), while feed forward motifs with SUM configuration show delay after signal deactivation (10). Feedback loops can lead to bistable behavior (18,20,21,(26)(27)(28), oscillations (12,19,(29)(30)(31)(32), signal delay (14,33), and can filter noise (29). Single-input modules (SIMs) (18) are sets of operons that are controlled by a single transcription factor that coordinates the activity of all the operons. This configuration can be used for signal sorting and output synchronization (13,14) (See Table 1 for summary and comparison between the functional capabilities of the various motifs). Bifans, which have been less well studied, were shown to be highly dependent on the gating mechanisms used (i.e. AND or OR) (22). However, the functional characteristics of bifans have not been studied in depth. Since the bifan consists of multiple activation routes for each target, a reasonable hypothesis would be that bifans function is similar to those of the feed-forward loop motif. Stability analysis of all possible small-size motifs identified classes of motifs, where the bifan motif was found to belong to Class I of Structural Stability Score (SSS). Motifs in this class have no loops and are shown to be most stable, while motifs with feedback loops are are least stable (23). This analysis implied that bifan motifs are expected to have stable dynamics.
The bifan motif is the most statistically over-represented network motif in signaling (9,11) and gene regulatory networks (5,22). The configuration of the bifan motif includes two source nodes directly cross-regulating two target nodes (shaded area in Fig. 1a). When considering the type of nodes and type of links making up each bifan, the bifan motif consisting of two protein kinases each phosphorylating and activating two transcription factors was found to be highly over-represented in a mammalian neuronal intracellular signaling network we constructed from literature (11). Hence, in this study, we have investigated the dynamical properties of a cell signaling bifan network motif made of two protein kinases regulating the activity of two transcription factors. Using a system of coupled ODEs we determined the intrinsic capabilities of this bifan motif.
Mitogen-activated protein kinase p38α and c-Jun N-terminal kinase-1 (JNK) are two protein kinases that phosphorylate and activate the cAMP-dependent Activating transcription factor-2 (ATF2) and ETS domain protein Elk-1 transcription factors. Transcription factors, when phosphorylated by protein kinases, become activated, and often form homodimers and/or hetrodimers that participate in dynamic nuclear complexes (34) and bind DNA promoter sequences to regulate transcription. ATF2 belongs to a family of transcription factors that can homodimerize or hetrodimerize with c-Fos, FosB, Fra1 and Fra2 or c-Jun, JunB, and JunD family members (35). As dimers, these transcription factors bind to specific promoter sites such as the AP1 consensus sequence TGAC/GTC/AA (36). When these dimers bind to the promoter they enable the assembly a complex that allows for DNA remodeling and initiation of transcription (37). Elk-1 forms homodimers and hetrodimers with TCF family members to bind to specific promoter elements such as SRF (38,39). JNK1 and p38α are protein kinases that target and phosphorylate Elk-1 at two different domains (40) and are also known to phosphorylate ATF2 (41,42) shown, for example, in response to UV irradiation and in neuroblastoma cells (43).
The bifan motif we have investigated in this study is fully coherent, where all links are positive (activating). A previous report on bifan motifs considered dynamics of bifans with both positive and negative links to analyze various
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