Living cells display complex signal processing behaviors, many of which are mediated by networks of proteins specialized for signal transduction. Here we focus on the question of how the remarkably diverse array of eukaryotic signaling circuits may have evolved. Many of the mechanisms that connect signaling proteins into networks are highly modular: The core catalytic activity of a signaling protein is physically and functionally separable from molecular domains or motifs that determine its linkage to both inputs and outputs. This high degree of modularity may make these systems more evolvable-in principle, novel circuits, and therefore highly innovative regulatory behaviors, can arise from relatively simple genetic events such as recombination, deletion, or insertion. In support of this hypothesis, recent studies show that such modular systems can be exploited to engineer nonnatural signaling proteins and pathways with novel behavior.

Many eukaryotic signaling proteins are composed of simple modular binding domains, yet they can display sophisticated behaviors such as allosteric gating and multi-input signal integration, properties essential for complex cellular circuits. To understand how such behavior can emerge from combinations of simple domains, we engineered variants of the actin regulatory protein N-WASP (neuronal Wiskott-Aldrich syndrome protein) in which the "output" domain of N-WASP was recombined with heterologous autoinhibitory "input" domains. Synthetic switch proteins were created with diverse gating behaviors in response to nonphysiological inputs. Thus, this type of modular framework can facilitate the evolution or engineering of cellular signaling circuits.

Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase.

At least 30% of human proteins are thought to contain intrinsically disordered regions, which lack stable structural conformation. Despite lacking enzymatic functions and having few protein domains, disordered regions are functionally important for protein regulation and contain short linear motifs (short peptide sequences involved in protein-protein interactions), but in most disordered regions, the functional amino acid residues remain unknown. We searched for evolutionarily conserved sequences within disordered regions according to the hypothesis that conservation would indicate functional residues. Using a phylogenetic hidden Markov model (phylo-HMM), we made accurate, specific predictions of functional elements in disordered regions even when these elements are only two or three amino acids long. Among the conserved sequences that we identified were previously known and newly identified short linear motifs, and we experimentally verified key examples, including a motif that may mediate interaction between protein kinase Cbk1 and its substrates. We also observed that hub proteins, which interact with many partners in a protein interaction network, are highly enriched in these conserved sequences. Our analysis enabled the systematic identification of the functional residues in disordered regions and suggested that at least 5% of amino acids in disordered regions are important for function.

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